KR20100028031A - Methods for improving protein properties - Google Patents

Abstract

The present invention provides methods for engineering proteins to optimize their performance under certain environmental conditions of interest. In some embodiments, the present invention provides methods for engineering enzymes to optimize their catalytic activity under particular environmental conditions. In some preferred embodiments, the present invention provides methods for engineering enzymes to optimize their catalytic activity and/or stability under adverse environmental conditions. In some preferred embodiments, the present invention provides methods for engineering enzymes to optimize their storage stability, particularly under adverse environmental conditions. In some preferred embodiments, the present invention provides methods for altering the net surface charge and/or surface charge distribution of enzymes (e.g., metalloproteases) to obtain enzyme variants that demonstrate improved performance and/or stability in detergent formulations as compared to the starting or parent enzyme.

Description

How to improve protein properties {METHODS FOR IMPROVING PROTEIN PROPERTIES}

Cross Reference to Related Applications

The present invention claims priority to US Provisional Serial Nos. 60 / 933,307, 60 / 933,331 and 60 / 933,312, filed June 6, 2007, which are incorporated by reference in their entirety.

The present invention provides a method of engineering a protein such that the performance of the protein is optimized under specific environmental conditions of interest. In some embodiments, the present invention provides a method of engineering an enzyme to optimize the catalytic activity of the enzyme under certain environmental conditions. In some preferred embodiments, the present invention provides a method of engineering an enzyme such that the catalytic activity and / or stability of the enzyme under opposing environmental conditions is optimized. In some preferred embodiments, the present invention provides a method of engineering an enzyme such that the storage stability of the enzyme is optimized under particularly opposing environmental conditions. In some preferred embodiments, the present invention provides a net surface charge and / or surface of an enzyme (eg metalloprotease) such that an enzyme variant is obtained that exhibits improved performance and / or stability in detergent formulations as compared to the starting or parent enzyme. Provides a way to alter the charge distribution.

Outside of his natural environment, the properties of protein functions are often suboptimal. For example, enzymes (eg proteases, lipases, amylases, cellulases, etc.) are often used to clean contaminants of fibers in laundry detergents, which typically comprise complex combinations of active ingredients. In fact, most cleaning products include surfactant systems, bleaches, wash builders, suds suppressors, soil-suspending agents, antifouling agents, fluorescent brighteners, softeners, dispersants, dye transfers. Inhibitory compounds, abrasives, fungicides and fragrances as well as cleaning enzymes. Thus, despite the complexity of current detergents, there are many contaminants that are difficult to remove completely, in part due to suboptimal enzyme performance. Despite much research in enzyme development, there is a need in the art for methods of engineering proteins for specific uses and conditions. Indeed, there is a need in the art for methods of quickly and systematically tailoring other electrostatic properties to optimize their performance in commercial applications. In particular, but not limited to, lipase, amylase, cutinase, mannanase, acid reduction enzymes, cellulase, pectinase, proteases and other enzymes to provide improved activity, stability and solubility in cleaning solutions. There is a need in the art for methods of engineering industrially useful enzymes.

Summary of the Invention

The present invention provides a method of engineering a protein such that the performance of the protein is optimized under certain environmental conditions of interest. In some embodiments, the present invention provides a method of engineering an enzyme to optimize the catalytic activity of the enzyme under certain environmental conditions. In some preferred embodiments, the present invention provides a method of engineering an enzyme to optimize the catalytic activity and / or stability of the enzyme under opposing environmental conditions. In some preferred embodiments, the present invention provides a method of engineering an enzyme such that the storage stability of the enzyme is optimized under particularly opposing environmental conditions. In some preferred embodiments, the present invention provides a net surface charge and / or an enzyme (eg metalloprotease) such that an enzyme variant is obtained that exhibits improved performance and / or stability in detergent formulations as compared to the starting or parent enzyme. It provides a method of changing the surface charge distribution.

The present invention provides a method of producing an improved protein variant, comprising: testing a plurality of single-substituted protein variants in a first test of a first property and a second test of a second property, Wherein the property of the parent protein is given a value of 1.0 in each test, the advantageous first or second property has a value greater than 1.0, the excessively disadvantageous first or second property is less than about 0.80, or some preferred embodiments Having a value of less than about 0.60; Identifying a substitution in one or more of the mono-substituted protein variants that are associated with the advantageous first property and not excessively disadvantageous the second property; Identifying a substitution in one or more of the mono-substituted protein variants associated with the second advantageous property and not associated with the overly adverse first property; Introducing a substitution from the previous step into the protein to obtain a multi-substituted protein variant. In some embodiments, the method further comprises testing the multi-substituted protein in the first test and the second test, wherein the improved protein variant has a value greater than 1.0 in both the first and second tests. Or a value greater than 1.0 in the first test and a value between 0.80 and 1.0 in the second test. In some additional embodiments, the method further comprises generating an improved protein variant (s). In some embodiments, the first and second characteristics are negatively correlated. In some further embodiments, the advantageous first or second property has a value greater than about 1.2. In some further embodiments, the first and second characteristics that are excessively disadvantageous have a value of less than about 0.40. In some preferred embodiments, the first property is stability and the second property is wash performance. In some particularly preferred embodiments, stability includes stability in detergents and cleaning performance comprises blood milk ink (BMI) cleaning performance in detergents. In some further preferred embodiments, the protein is a neutral metalloprotease. In some additional embodiments, the parent protein is a wild type mature form of the neutral metalloprotease, while in other embodiments the variant is derived from the neutral metalloprotease of the family Bacillaceae. In some particularly preferred embodiments, the variants are derived from neutral metalloproteases of the genus Bacillus. In still further embodiments the wash performance is tested in a liquid detergent composition or powder having a pH of 5 to 12.0. In some further embodiments the wash performance is tested in a cold water liquid detergent having a basic pH. In still further embodiments, at least one of the substitutions comprises a net charge change of 0, -1, or -2 relative to the parent neutral metalloprotease, while in some alternative embodiments at least one of the substitutions is parent neutral metalloprotease With a net charge change of +1 or +2. Since any suitable order is used in the present invention, it is not intended that the steps be limited to the exact order listed above. In some preferred embodiments, the improved protease variants have a net charge change of +1 or +2 relative to the parent neutral metalloprotease. In still further embodiments, the substitution is in position at the parent neutral metalloprotease having more than about 50% solvent accessible surface (SAS). In still further embodiments, the one or more positions in the parent neutral metalloprotease are positions having at least about 65% solvent accessible surface (SAS).

The present invention provides a method of producing an improved protease variant, comprising: testing a plurality of single-substituted protease variants in a first test of a first property and a second test of a second property, Wherein the property of the parent protease is given a value of 1.0 in each test, the advantageous first or second property has a value greater than 1.0, the excessively unfavorable first or second property is less than about 0.80, or some preferred embodiment Example has a value of less than about 0.60; Identifying a substitution in one or more of the mono-substituted protease variants that are associated with the advantageous first property and not excessively disadvantageous the second property; Identifying a substitution in one or more of the mono-substituted protease variants associated with the second advantageous property and not associated with the first disadvantageous property; Introducing a substitution from the previous step into the protease to obtain a multi-substituted protease variant. In some embodiments, the method further comprises testing the multi-substituted protease variants in the first test and the second test, wherein the improved protein variant is greater than 1.0 in both the first and second tests. Value, or greater than 1.0 in the first test and 0.80 to 1.0 in the second test. In some additional embodiments, the method also includes generating an improved protease variant (s). In some embodiments, the first and second characteristics are inversely related. In some further embodiments, the advantageous first or second property has a value greater than about 1.2. In some further embodiments, the first or second characteristic that is excessively disadvantageous has a value of less than about 0.40. In some preferred embodiments, the first property is stability and the second property is wash performance. In some particularly preferred embodiments, stability includes stability in detergents, and cleaning performance comprises washing performance of blood milk ink (BMI) in detergents. In some further preferred embodiments, the protease is a neutral metalloprotease. In some additional embodiments, the parent protease is a wild type mature form of neutral metalloprotease, while in other embodiments the variant is derived from the neutral metalloprotease of the family of Bacillaceae. In some particularly preferred embodiments, the variants are derived from neutral metalloproteases of the genus Bacillus. In still further embodiments the wash performance is tested in a liquid detergent composition or powder having a pH of 5 to 12.0. In some additional embodiments, the wash performance is tested in cold water liquid detergents with basic pH. In still further embodiments, one or more of the substitutions comprises a net charge change of 0, -1 or -2 relative to the parent neutral metalloprotease, while in some alternative embodiments one or more of the substitutions are parent neutral metalloprotease With a net charge change of +1 or +2. Since any suitable order is used in the present invention, it is not intended that the steps be limited to the exact order listed above. In some preferred embodiments, the improved protease variants have a net charge change of +1 or +2 relative to the parent neutral metalloprotease. In still further embodiments, the substitution is in position at the parent neutral metalloprotease having more than about 50% solvent accessible surface (SAS). In still further embodiments, the one or more positions in the parent neutral metalloprotease are positions having greater than about 65% solvent accessible surface (SAS). The invention also provides for multiple substituted proteins produced using the methods described herein. In some preferred embodiments, the present invention provides neutral metalloprotease variants produced by the methods described herein. In some particularly preferred embodiments, the invention provides protease variants comprising a substitution at a residue position corresponding to residue position 83 of the Bacillus neutral metalloprotease described in SEQ ID NO: 3. In some further preferred embodiments, the substitutions include L83K substitutions. Also provided are NprE variants comprising a combination of substitutions selected from the group consisting of:

i) 4K-45K-50R-54K-59K-90K-129I-138L-179P-190L-199E-214Q-220E-244S-265P-269H-285R-296E; 45K-50R-59K-90K-129I-138L-179P-190L-199E-214Q-220E-244S-265P-285R; 45K-59K-90K-129I-138L-179P-190L-199E-214Q-220E-265P-285R; And 59K-90K-129I-179P-190L-199E-214Q-220E-265P-285R.

The invention also provides an isolated polynucleotide encoding a protease variant described herein. In addition, the present invention provides expression vectors comprising the polynucleotides described herein in operable combinations with a promoter. The invention also provides host cells transformed with the expression vector (s) provided herein. The invention also provides cleaning compositions comprising protease variants produced using the methods of the invention.

The present invention provides an improved method for producing protein variants comprising the following steps:

a) testing a plurality of mono-substituted protein variants in a first test of a first property and a second test of a second property, wherein the properties of the parent protein are given a value of 1.0 in each test, The first or second property has a value of greater than 1.0 and the first or second property that is excessively disadvantageous has a value of less than about 0.80; b) identifying a substitution in one or more of the mono-substituted protein variants that are associated with the first advantageous property and not the second property that are excessively disadvantageous; c) identifying a substitution in at least one of the mono-substituted protein variants associated with the second advantageous property and not overly unfavorable the first property; d) introducing the substitution from step b) and the substitution from c) into the protein to obtain a multi-substituted protein variant. In some embodiments, the method further comprises the following steps: e) testing the multi-substituted protein variants in the first test and the second test, wherein the improved protein variant is present in both the first and second tests. To achieve a value greater than 1.0, or to achieve a value of greater than 1.0 in a first test and a value of 0.80 to 1.0 in a second test.

In some embodiments, the protein is an enzyme selected from the group consisting of proteases, amylases, cellulases, polyesterases, esterases, lipases, cutinases, pectinase, oxidases, transferases, catalases and alcalases. In some preferred embodiments, the enzyme is a protease or amylase. In some embodiments, the first and second properties of interest include two or more properties selected from the group consisting of: substrate binding, enzyme inhibition, expression, stability in detergents, thermal stability; Reaction rate; Degree of reaction; Thermal activity; Starch liquefaction; Biomass degradation, saccharification, ester hydrolysis, enzymatic bleaching, washing performance and fabric modification. In some particularly preferred embodiments, the method also comprises generating an improved protein variant. In some embodiments, the first and second characteristics are inversely related. Advantageous first or second properties in some embodiments of the present invention have a value of greater than about 1.2 and / or excessively disadvantageous first or second properties have a value of less than about 0.60. In some preferred embodiments, the first property is stability and the second property is wash performance. In a subset of these embodiments, stability includes stability in detergents and cleaning performance includes blood milk ink (BMI) cleaning performance in detergents. In some embodiments, the first property is protein expression and the second property is enzymatic activity. In a subset of these embodiments, enzymatic activity includes rice starch washing performance in detergents. In some preferred embodiments, the protease is selected from the group consisting of neutral metalloproteases, serine proteases and subtilisin. In a subset of these embodiments, the neutral metalloprotease is a neutral metalloprotease of the family of Bacillaceae. In some exemplary embodiments, the neutral metalloprotease is of the genus Bacillus (eg, B. subtilis NprE). In a further embodiment, the amylase is alpha amylase of the family of Bacillaceae. In an exemplary embodiment, the alpha amylase is of the genus Bacillus (eg B. stearothermophilus Amys). In some preferred embodiments, wash performance is tested in liquid detergent compositions or powders having a pH of 5 to 12.0, and / or cold water liquid detergents having a basic pH. In some embodiments, one or more of the substitutions comprise a net charge change of 0, -1 or -2 with respect to the parent enzyme. In some embodiments, one or more of the substitutions comprise a net charge change of +1 or +2 relative to the parent enzyme. In some preferred embodiments, one or more of the substitutions have two or more substitutions, the first having a net charge change of 0, -1 or -2 with respect to the parent enzyme; The second involves having a net charge change of +1 or +2 relative to the parent enzyme. In some embodiments, one or more substitutions are from 1 to 20 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In some embodiments, the improved enzyme variant has a net charge change of -2, -1, 0, +1 or +2 with respect to the parent enzyme. In some embodiments, the substitution is in position at the parent enzyme having more than about 25%, more than about 50%, or more than about 65% solvent accessible surface (SAS). Also provided by the present invention are isolated polynucleotides encoding the enzyme variants described herein. In a further embodiment the invention provides expression vectors comprising polynucleotides in operable combinations with a promoter. In some embodiments, a host cell comprising an expression vector is provided. In a further embodiment, a cleaning composition is provided comprising an enzyme variant produced using the method of the invention.

The present invention provides an improved method for producing protein variants, comprising the following steps in a viable order: a) a plurality of single-substituted protein variants in a first test of a first property and a second test of a second property. Testing, wherein the properties of the parent protein are given a value of 1.0 in each test, wherein the advantageous first or second property has a value of greater than 1.0 and the excessively unfavorable first or second property has a value of less than about 0.80 Having; b) identifying a substitution in one or more of the mono-substituted protein variants that are associated with the first advantageous property and not the second property that are excessively disadvantageous; c) identifying a substitution in at least one of the mono-substituted protein variants associated with the second advantageous property and not overly unfavorable the first property; d) introducing the substitutions from steps b) and c) into the protein to obtain multi-substituted protein variants, wherein the multi-substituted protein variants are improved protein variants. In some preferred embodiments, the method further comprises the steps of: e) testing the multi-substituted protein variants in the first and second trials, wherein the improved protein variants are tested in the first and second trials. A value greater than 1.0 is achieved in all, or a value greater than 1.0 in the first test and a value of 0.80 to 1.0 in the second test. In some additional embodiments, the parent protein is an enzyme, wherein the improved protein variants are enzymes. In some additional embodiments, the enzyme is selected from proteases, amylases, cellulases, polyesterases, esterases, lipases, cutinases, pectinase, oxidases, transferases and catalases. In some particularly preferred embodiments, the enzyme is a protease or amylase. In some additional embodiments, the first and second properties of interest are substrate binding, enzyme inhibition, expression, stability in detergents, thermal stability, reaction rate, degree of reaction, thermal activity, starch liquefaction, biomass degradation, glycosylation, esters At least two of the group consisting of hydrolysis, enzymatic bleaching, washing performance and fabric modification. In some further embodiments, the method also includes generating an improved protein variant. In some additional embodiments, the first and second characteristics are inversely related. In some particularly preferred embodiments, the advantageous first or second property has a value of greater than about 1.2. In some further preferred embodiments, the first or second characteristic that is excessively disadvantageous has a value of less than about 0.60. In some further embodiments, the first or second characteristic that is excessively disadvantageous has a value of less than about 0.40. In some preferred embodiments, the first property is stability and the second property is wash performance. In some particularly preferred embodiments, stability includes stability in the detergent composition and cleaning performance comprises blood milk ink (BMI) cleaning performance. In some further embodiments, the wash performance is tested in a liquid detergent composition or powder having a pH of about 5 to about 12. In some further preferred embodiments, the wash performance is tested in a cold water liquid detergent comprising a basic pH. In some additional embodiments, the first property is protein expression and the second property is enzymatic activity. In some additional embodiments, the protease is selected from neutral metalloproteases and serine proteases. In some particularly preferred embodiments, the serine protease is subtilisin. In some additional embodiments, the neutral metalloprotease is a neutral metalloprotease obtained from a member of the family of Bacillaceae. In some alternative embodiments, the amylase is an alpha amylase obtained from a member of the family Basilase. In some additional embodiments, one or more of the substitutions comprise a net charge change of 0, -1 or -2 with respect to the parent enzyme. In some alternative embodiments, one or more of the substitutions comprise a net inversion change of +1 or +2 with respect to the parent enzyme. In some further embodiments, one or more of the substitutions comprise a net charge change of 0, -1 or -2 with respect to the parent enzyme. In some additional alternative embodiments, one or more of the substitutions comprises a net charge change of +1 or +2 relative to the parent enzyme. In some further embodiments, the improved enzyme variant has a net charge change of +1 or +2 relative to the parent enzyme. In some alternative embodiments, the substitution is in position at the parent enzyme having more than about 25% solvent accessible surface (SAS). In some preferred embodiments, the substitution is in position at the parent enzyme having more than about 50% or more than about 65% solvent accessible surface (SAS). In some particularly preferred embodiments, the parent enzyme is a wild type enzyme.

The invention also provides a cleaning composition comprising improved protein variants produced according to the methods described herein.

The invention also provides isolated neutral metalloprotease variants having an amino acid sequence comprising one or more substitutions of amino acids made at positions equivalent to those in the neutral metalloprotease comprising the amino acid sequence set forth in SEQ ID NO: 3. . In some preferred embodiments, one or more substitutions are made at positions equivalent to position 83 of the amino acid sequence set forth in SEQ ID NO: 3. In some particularly preferred embodiments, the substitution is L83K.

1A shows relative specific BODIPY-starch hydrolysis activity versus shake tube expression for AmyS-S242Q Combination Charge Library (CCL).

1B shows relative rice starch Microswatch cleaning activity versus shake tube expression in TIDE 2 × for AmyS-S242Q CCL.

2A shows relative shake tube expression versus relative net charge change for AmyS-S242Q CCL.

2B shows the relative BODIPY-starch hydrolysis activity versus relative net charge change for AmyS-S242Q CCL.

3A shows relative shake tube expression versus relative net charge change for AmyS-S242Q CCL.

3B shows rice starch microsample cleaning activity versus relative net charge change for AmyS-S242Q CCL.

General description of the invention

The present invention provides a method of engineering a protein such that performance is optimized under specific environmental conditions of interest. In some embodiments, the present invention provides a method of engineering an enzyme to optimize catalytic activity under certain environmental conditions. In some preferred embodiments, the present invention provides a method of engineering an enzyme to optimize catalyst activity and / or stability under opposing environmental conditions. In some preferred embodiments, the present invention provides a method of engineering an enzyme to optimize storage stability under particularly opposing environmental conditions. In some preferred embodiments, the present invention provides a net surface charge and / or an enzyme (eg metalloprotease) such that an enzyme is obtained that exhibits improved performance and / or stability in detergent formulations as compared to the starting or parent enzyme. It provides a method of changing the surface charge distribution.

Protease subtilisin is the main enzyme used in laundry detergents and is probably the most widely used enzyme in the world. It has been noted that surface electrostatic effects can modulate the catalytic activity of subtilisin (see, eg, Russell and Fersht, Nature 328: 496-500 [1987]).

More recently, it has been observed that mutations, including changing the net charge of subtilisin, have a dramatic effect on the washing performance of detergents (see, eg EP 0 479 870 B1). This beneficial effect was considered to be the result of shifting the pi (isoelectric point) of the subtilisin to the pH of the cleaning liquid. However, in subsequent studies, the conclusions have not always been applicable (see eg US Pat. No. 6,673,590 B1). As indicated in this patent, the effect of charge mutations on subtilisin is highly dependent on detergent concentrations, which lower the pi of the parent subtilisin to provide more effective enzymes at lower detergent concentrations, The pI is raised to provide more effective enzymes at higher detergent concentrations. This is quite useful because the detergent concentrations in the cleaning liquids vary widely around the world. Thus, it will be apparent to those skilled in the art that there is an optimal pi for the wash performance of subtilisin, which depends on the pH and detergent concentration of the wash liquid. Further efforts have been described to improve the activity of subtilisin in laundry detergents (see US Patent Publication 2005/0221461). Surprisingly, it was found that subtilisin variants with the same net electrostatic charge as the parent subtilisin have increased washing performance at both high and low detergent concentration wash conditions.

Unless otherwise indicated, the practice of the present invention all encompasses conventional techniques commonly used in protein engineering, molecular biology, microbiology, and recombinant DNA within the art. Such techniques are those known to those skilled in the art and are described in many literatures and reference articles known to those skilled in the art. All patents, patent applications, articles, and publications mentioned above and below herein are expressly incorporated herein by reference for their valid teachings relating to the methods and compositions provided by the present invention.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are described in more detail with reference to the specification as a whole.

Also, as used herein, the singular includes the plural reference unless the context clearly indicates otherwise. The numerical range includes the numbers that define the range. Unless otherwise indicated, nucleic acids are indicated from left to right in the 5 'to 3' direction; Amino acid sequences are indicated from left to right, respectively, in the amino to carboxy direction. It is to be understood that the invention is not so limited, as the particular methodologies, protocols and reagents described may vary depending on the context used by those skilled in the art.

All maximum numerical limits given throughout this specification are intended to include all lower numerical limits, as if the lower numerical limits were expressly written herein. All minimum numerical limits given throughout this specification will include all higher numerical limits as if the higher numerical limits were clearly written herein. All numerical ranges given throughout this specification will include all narrower numerical ranges falling within such broader numerical ranges as if all narrower numerical ranges were expressly written herein.

Moreover, the headings provided herein are not intended to limit the various aspects or embodiments of the invention, which may be given by reference to the specification as a whole. Accordingly, the terms defined immediately below are defined in more detail by reference to the specification as a whole. Nevertheless, in order to facilitate understanding of the present invention, many terms are defined below.

Justice

As used herein, the terms “protease,” and “proteolytic activity” refer to proteins or peptides that exhibit the ability to hydrolyze a peptide or substrate with peptide linkages. Many well known procedures exist for the determination of proteolytic activity (Kalisz, "Microbial Proteinase," In: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology , [1988]. For example, proteolytic activity can be confirmed by comparative assays that analyze the ability of each protease to hydrolyze conventional substrates. Exemplary substrates useful for the analysis of protease or proteolytic activity include di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111). Include but are not limited to. Colorimetric assays using such substrates are well known in the art (see eg WO 99/34011; and US Pat. No. 6,376,450). pNA assays (see, eg, Del Mar et al., Anal. Biochem., 99: 316-320 [1979]) can also be used to determine active enzyme concentrations for fractions collected during gradient elution. The assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA), a soluble synthetic substrate. The rate of formation of yellowish tint from the hydrolysis reaction is measured at 410 nm with a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nm can be used to determine total protein concentration. The active enzyme / total-protein ratio provides the enzyme purity.

As used herein, the terms “ASP protease,” “Asp protease,” and “Asp” refer to the serine proteases described herein and are described in US Patent Application Serial No. 10 / 576,331. In some preferred embodiments, the Asp protease is a protease designed herein as the 69B4 protease obtained from Cellulomonas strain 69B4. Thus, in a preferred embodiment, the term “69B4 protease” refers to a naturally occurring mature protease derived from Cellulomonas strain 69B4 (DSM 16035). In alternative embodiments, the present invention provides a portion of an ASP protease.

The term “cellulomonas protease homologue” refers to a naturally occurring protease having an amino acid sequence substantially identical to a mature protease from Cellulomonas strain 69B4, or a polynucleotide sequence encoding said naturally occurring protease, said protease Retains the functional characteristics of the serine protease encoded by such nucleic acid. In some embodiments, the protease homologues are called "cellulomonadin".

As used herein, the terms "ASP variant," "ASP protease variant," and "69B protease variant" are particularly similar in function to wild type ASP, but have mutations in their amino acid sequences that result in sequence with the wild type protease. It is used to refer to proteases that are different.

As used herein, “Cellulomonas ssp.” Refers to all species within the genus “Cellulomonas”, which refers to the family Cellulomonadaceae, microcosinea. It is a Gram-positive bacterium classified into members of Suborder Micrococcineae, Order Actinomycetales, and Class Actinobacteria. It is recognized that there is a continuous taxonomic reorganization of the genus Cellulomonas. Thus, genus names are intended to include reclassified species names.

As used herein, "Streptomyces ssp." Refers to all species within the genus "Streptomyces", which refers to the family Streptomycetaceae, Streptomycinia. It is a Gram-positive bacterium classified into members of Suborder Streptomycineae, Order Actinomycetales, and Class Actinobacteria. It is recognized that there is a continuous taxonomic reorganization of the genus Streptomyces. Thus, genus names are intended to include reclassified species names.

As used herein, “genus Bacillus” includes B. subtilis, B. licheniformis, B. lentus, B. B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausi B. clausii), B. halodurans, B. megaterium, B. coagulans, B. circulans, B. All species within the genus "Bacillus" are included, as known to those of skill in the art, including but not limited to B. lautus, and B. thuringiensis. It is recognized that there is a continuous taxonomic reorganization of the genus Bacillus. Thus, the generic name includes reclassified species, including but not limited to, such organisms as B. stearothermophilus, now referred to as "Geobacillus stearothermophilus." It is intended. The production of resistant endospores in the presence of oxygen is characterized by the fact that the present names are also known as Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus. ), Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Thermobacillus, Uribacillus and Ureibacillus Although applied to Virgibacillus, it is considered to define the characteristics of the genus Bacillus.

As used interchangeably herein, the terms “polynucleotide” and “nucleic acid” refer to polymer forms (ribonucleotides or deoxyribonucleotides) of nucleotides of any length. The term includes single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids, or purine and pyrimidine bases, or other natural, chemically, or chemically modified, non-natural or inducible. Polymers comprising, but not limited to, nucleotide bases. The following are non-limiting examples of polynucleotides: genes, gene segments, chromosomal segments, ESTs, exons, introns, mRNAs, tRNAs, rRNAs, ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, Isolated DNA of any sequence, Isolated RNA of any sequence, nucleic acid probes and primers. In some embodiments, polynucleotides include modified nucleotides such as methylated nucleotides and nucleotide analogues, uracil, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. In alternative embodiments, the sequence of nucleotides is interrupted by non-nucleotide components.

As used herein, the terms “DNA construct” and “transformation DNA” are used interchangeably to refer to DNA used to introduce a sequence into a host cell or organism. DNA can be generated in vitro by PCR or any other suitable technique (s) known to those skilled in the art. In a particularly preferred embodiment, the DNA construct comprises a sequence of interest (eg, an incoming sequence). In some embodiments, the sequence is operably linked to additional elements, such as regulatory elements (eg, promoters, etc.). The DNA construct may further comprise a selectable marker. It may further comprise a successor sequence on the side of the homology box. In further embodiments, the transforming DNA comprises other nonhomologous sequences added at the ends (eg, stuffer sequences or flanks). In some embodiments, the ends of the successor sequences are tight so that the transforming DNA forms a closed ring. Transformation sequences may be wild type, mutant, or may be modified. In some embodiments, the DNA construct comprises a sequence homologous to a host cell chromosome. In other embodiments, the DNA construct comprises a nonhomologous sequence. Once the DNA construct is assembled in vitro, it can be used to: 1) insert the heterologous sequence into the desired target sequence of the host cell; And / or 2) mutating the site of the host cell chromosome (ie, replacing the endogenous sequence with a heterologous sequence), and / or 3) eliminating the target gene; And / or introducing a replication plasmid into the host.

As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct that has been produced recombinantly or synthetically, as a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plasmid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector comprises, among other sequences, the nucleic acid sequence and promoter to be transcribed. In a preferred embodiment, the expression vector has the ability to incorporate and express heterologous DNA fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of the appropriate expression vector is within the knowledge of those skilled in the art. The term "expression cassette" is used herein interchangeably with "DNA construct" and its grammatical equivalents. Selection of the appropriate expression vector is within the knowledge of those skilled in the art.

As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes and the like. In some embodiments, the polynucleotide construct comprises a DNA sequence encoding a protease (eg, precursor or mature protease), the sequence comprising a suitable prosequence (e.g., that can affect the expression of DNA in a suitable host). For example, secretion, etc.).

As used herein, the term “plasmid” refers to a cyclic double-stranded (ds) DNA construct used as a cloning vector, which forms an extrachromosomal autologous genetic element in some eukaryotic or prokaryotic cells, Or incorporated into the host chromosome.

As used herein, in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence to the cell. Such transduction methods include, but are not limited to, plasma fusion, transfection, transformation, conjugation and transduction (eg, Ferrari et al., “Genetics,“ in Hardwood et al. (Eds.), Bacillus , Plenum Publishing Corp., pages 57-72 [1989]).

As used herein, the terms “transformed” and “stablely transformed” refer to a cell having a unique (heterologous) polynucleotide sequence incorporated into the genome of the cell, or that is maintained for two or more generations. Cells as episomal plasmids are shown.

As used herein, the term “selectable marker-encoding nucleotide sequence” refers to a nucleotide sequence that can be expressed in a host cell, wherein expression of the selectable marker is in the presence of a corresponding selection agent or in the absence of essential nutrients. It is endowed to cells containing the expressed genes that can grow.

As used herein, the terms “selectable marker” and “selective marker” refer to a nucleic acid (eg, a gene) that can be expressed in a host cell that allows for easy selection of the host containing the vector. Examples of such selectable markers include but are not limited to antibiotics. Thus, the term "selectable marker" refers to a gene that provides an indication that the host cell has taken successor DNA of interest or that some other reaction has occurred. Typically, selectable markers are genes that confer metabolic benefits or antibiotic resistance to a host cell so that cells containing exogenous DNA can be distinguished from cells that do not receive any exogenous sequence during transformation. A "residing selectable marker" is a marker located on the chromosome of the microorganism to be transformed. The resident selectable marker encodes a gene that is different from the selectable marker on the transforming DNA construct. Selective markers are well known to those skilled in the art. As indicated above, preferably the marker is an antibiotic resistance marker (eg, amp ^R ; phleo ^R ; spec ^R ; kan ^R ; ery ^R ; tet ^R ; cmp ^R ; and neo ^R (eg, Guerot- Fleury, Gene, 167: 335-337 (1995); Palmeros et al., Gene 247: 255-264 [2000]; and Trieu-Cuot et al., Gene, 23: 331-341, [1983]. Other markers useful in accordance with the present invention include nutritional utero markers such as tryptophan; And detection markers such as β-galactosidase.

As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct transcription of downstream genes. In a preferred embodiment, the promoter is suitable for the host cell in which the target gene is expressed. A promoter, along with other transcriptional and translational regulatory nucleic acid sequences (also called "regulatory sequences"), is required for the expression of a given gene. In general, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancers or active sequences.

A nucleic acid is “operably linked” when it is positioned to have a functional relationship with another nucleic acid sequence. For example, a DNA encoding secretory leader (ie, signal peptide) is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it is located to facilitate translation. In general, "operably linked" means that the DNA sequences to which they are linked are contiguous, and in the case of a secretory leader, are contiguous and in the reading phase. However, enhancers do not have to be contiguous. Linking is by linkage at conventional restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

The term “gene” as used herein refers to a polynucleotide (eg, a DNA segment) that encodes a polypeptide and includes an intervening sequence (intron) between individual coding segments (exons) as well as sites before and after coding sites. Indicates.

As used herein, "homologous genes" refer to pairs of genes that differ from one another, but usually from related species, which homologous genes correspond to one another and are identical or very similar to one another. The term refers to genes isolated by speciation (ie, development of new species) (eg orthologous genes) as well as genes isolated by genetic replication (eg paralogous). Gene).

As used herein, “orthologs” and “ortholog genes” refer to genes of different species that have evolved from a common ancestor gene (ie, homologous gene) by speciation. Typically, orthologs retain the same function during evolution. Identification of orthologs is used to reliably predict gene function in a newly sequenced genome.

As used herein, "paralog" and "paralog gene" refer to genes involved by duplication in the genome. Orthologs retain the same function throughout the evolution process, while paralogs evolve new functions even though some functions are often related to the original. Examples of paralog genes include, but are not limited to, genes encoding trypsin, chymotrypsin, elastase, and thrombin, all of which are serine proteases and occur together in the same species.

As used herein, “homology” refers to sequence similarity or identity, with identity being preferred. The homology is measured using standard techniques known in the art (eg, Smith and Waterman, Adv. Appl. Math., 2: 482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48 : 443 [1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 [1988]; programs such as GAP, BESTFlT, FASTA, and TFASTA {Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI) And Devereux et al., Nucl.Acid Res., 12: 387-395 [1984]).

As used herein, “similar sequence” is a sequence whose function is essentially the same as a gene based on the parent gene (eg, cellulose monas strain 69B4 protease). In addition, the analogous gene is at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about the sequence of the parent gene. 90%, about 95%, about 97%, about 98%, about 99% or about 100% sequence identity. Alternatively, the analogous sequence has 70-100% alignment of the gene found at the parent gene (eg, Cellulomonas strain 69B4 protease) site, and / or has a parent gene (eg, cellulosic) At least 5 to 10 genes found at sites aligned with the genes in the chromosome containing Monas strain 69B4 chromosome. In further embodiments, more than one of the above properties is applied to the sequence. Analogous sequences are determined by known sequence alignment methods. A commonly used alignment method is BLAST, but there are other methods that can be used to align sequences, as shown above and below.

One example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from a group of related sequences using progressive, two-pair alignments. It can also draw a tree showing the clustering relationship used to make the alignment. PILEUP uses a simplification of Feng and Doolittle's incremental alignment method (Feng and Doolittle, J. Mol. Evol., 35: 351-360 [1987]). The method is similar to the method described by Higgins and Sharp (Higgins and Sharp, CABIOS 5: 151-153 [1989]). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap.

Another example of a useful algorithm is the BLAST algorithm described by Altschul et al. (Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Karlin et al., Proc. Natl. Acad. Sci., USA, 90: 5873-5787 (1993)). A particularly useful BLAST program is the WU-BLAST-2 program (see Altschul et al., Meth. Enzymol., 266: 460-480 [1996]). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set using the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. HSP S and HSP S2 parameters are It is a dynamic value and is built by the program itself depending on the composition of the particular database and the composition of the particular sequence from which the sequence of interest is searched. However, the value can be adjusted to increase the sensitivity. The% amino acid sequence identity value is determined by dividing the number of identical residues that match by the total number of residues of the "longer" sequence in the aligned site. A “longer” sequence is one with the most actual residue at the aligned site (ignoring the gap introduced by WU-Blast-2 to maximize the alignment score).

Thus, "% nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues of the starting sequence (ie, the sequence of interest). The preferred method uses the BLASTN module of WU-BLAST-2 to set the default parameters, setting the duplicate span and duplicate fraction to 1 and 0.125, respectively.

As used herein, the term “hybridization” refers to a method in which one strand of a nucleic acid binds to a complementary strand through base pairs, as is known in the art.

If the two sequences specifically hybridize to each other under medium to high stringent hybridization and wash conditions, the nucleic acid sequence is considered to be "selectively hybridizable" to the reference nucleic acid sequence. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm-5 ° C. (5 ° C. lower than the Tm of the probe); “High stringency” occurs at a temperature about 5-10 ° C. below the Tm; “Moderate stringency” occurs at a temperature about 10-20 ° C. below the Tm of the probe; "Low stringency" occurs at temperatures about 20-25 ° C. below the Tm. Functionally, maximum stringent conditions can be used to identify sequences with strict or near strict identity using hybridization probes; On the other hand, medium or low stringent hybridization can be used to identify or detect polynucleotide sequence homologs.

Medium and high stringent hybridization conditions are well known in the art. Examples of high stringent conditions are hybridization at about 42 ° C. in 50% formamide, 5X SSC, 5X Denhardt solution, 0.5% SDS and 100 μg / ml denatured carrier DNA, followed by 2X SSC and 0.5% SDS Washing twice at room temperature and further washing at 42 ° C., in 0.1 × SSC and 0.5% SDS. Examples of moderately stringent conditions include 20% formamide, 5X SSC (15O mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt solution, 10% dextran sulfate and 20 mg / In a solution containing ml denatured sheared salmon sperm DNA, incubate at 37 ° C. overnight, followed by washing the filter at about 37-50 ° C., 1 × SSC. Those skilled in the art know how to adjust the temperature, ionic strength, and the like necessary to adjust factors such as probe length and the like.

As used herein, “recombinant” includes those that refer to cells or vectors modified by the introduction of heterologous nucleic acid sequences, or cells derived from such modified cells. Thus, for example, a recombinant cell expresses a gene that is not found in the same form in its native (non-recombinant) form, or is expressed as a result of intentional human intervention or not at all, ie It expresses native genes that are otherwise abnormally expressed. The production of “recombinant,” “recombinantized,” and “recombined” nucleic acids is generally an assembly of two or more nucleic acid fragments, which assembly produces a chimeric gene.

In a preferred embodiment, the mutant DNA sequence is generated by site saturation mutagenesis in one or more codons. In another preferred embodiment, site saturation mutagenesis is performed for two or more codons. In further embodiments, the mutant DNA sequence is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90% with the wild type sequence, More than 95%, or more than 98% homology. In alternative embodiments, mutant DNA is generated in vivo using any known mutagenesis process such as irradiation, nitrosoguanidine, and the like. The desired DNA sequence is then isolated and used in the methods provided herein.

As used herein, the term “target sequence” refers to a DNA sequence in a host cell that encodes the sequence if it is desired to insert a successor sequence into the host cell genome. In some embodiments, the target sequence encodes a functional wild type gene or operon, while in other embodiments, the target sequence encodes a functional mutant gene or operon, or a non-functional gene or operon.

As used herein, “flanking sequence” refers to any sequence upstream or downstream of the sequence under discussion (eg, for gene ABC, gene B is the side of the A and C gene sequences In). In a preferred embodiment, the successor sequence is on the side of the homology box on each side. In another embodiment, the successor sequence and homology box comprise units flanking the stuffer sequence on each side. In some embodiments, flanking sequences exist only on a single side (3 'or 5'), but in preferred embodiments, the sequences are on each side of the flanking sequence. In some embodiments, flanking sequences exist only on a single side (3 'or 5'), while in preferred embodiments, the sequences are on each side of the flanking sequence.

As used herein, the term “stuffer sequence” refers to any external DNA (typically vector sequence) on the side of a homology box. However, the term includes any nonhomologous DNA sequence. Without wishing to be bound by theory, the stuffer sequence provides an insignificant target for the cell to initiate DNA acquisition.

As used herein, the terms “amplification” and “gene amplification” refer to a method in which a particular DNA sequence is overly replicated such that the amplified gene is present at a higher copy number than was originally present in the genome. In some embodiments, the selection of cells by growth in the presence of a drug (eg, inhibitor of an inhibitory enzyme) results in the amplification of an endogenous gene or the gene product encoding a gene product required for growth in the presence of the drug. Resulting in amplification, or both, of the exogenous (ie injected) sequence of encoding.

"Amplification" is a special case of nucleic acid replication associated with template specificity. This is in contrast to non-specific template replication (ie, replication that is template-dependent but not dependent on the specific template). Template specificity is here distinguished from replication fidelity (ie, synthesis of suitable polynucleotide sequences) and nucleotide (ribo- or deoxyribo-) specificities. Template specificity is often described in terms of “target” specificity. Target sequences are "targets" in that they are extracted from other nucleic acids. Amplification techniques were primarily designed for this extraction.

As used herein, the term "co-amplification" refers to an amplifiable marker along with other gene sequences (ie, including one or more non-selective genes, such as genes contained in an expression vector). Introduced into a single cell and application of appropriate selective pressure, the cells amplify both amplifiable markers and other, non-selective gene sequences. Amplifiable markers may be physically linked to other gene sequences or alternatively two separate DNA fragments, one containing amplifiable markers and the other containing non-selective markers, may be introduced into the same cell.

As used herein, the terms “amplifiable marker,” “amplifiable gene” and “amplification vector” refer to a vector or gene that encodes a gene that allows for amplification of the gene under appropriate growth conditions.

"Template specificity" is achieved with most amplification techniques by enzyme selection. Amplifying enzymes are enzymes that will process only nucleic acids of specific sequences in a heterogeneous mixture of nucleic acids under the conditions used. For example, for Qβ replicaases, MDV-1 RNA is a specific template for replicaases (see, eg, Kacian et al., Proc. Natl. Acad. Sci. USA 69: 3038 [1972]), and other nucleic acids. Is not replicated by the amplification enzyme. Similarly, in the case of T7 RNA polymerase, the amplification enzyme has strict specificity for its own promoter (see Chamberlin et al., Nature 228: 227 [1970]). In the case of T4 DNA ligase, the enzyme will not link two oligonucleotides or polynucleotides that have a mismatch between the oligonucleotide or polynucleotide substrate and the template at the junction (Wu and Wallace, Genomics 4: 560 [1989] ] Reference). Finally, Taq and Pfu polymerases are defined by primers as they are found to exhibit high specificity for the bound sequence in view of their ability to act at high temperatures; High temperatures result in thermodynamic conditions that favor primer hybridization with the target sequence and do not favor hybridization with non-target sequences.

As used herein, the term “amplifiable nucleic acid” refers to a nucleic acid that can be amplified by any amplification method. It is contemplated that an "amplifiable nucleic acid" will typically include a "sample template."

As used herein, the term “sample template” refers to a nucleic acid that originates from a sample analyzing the presence of “target” (defined below). In contrast, “background template” is used for nucleic acids other than sample templates that may or may not be present in a sample. Background templates are often inadvertent. This may be a residual result or may be due to the presence of nucleic acid contaminants to be purified and removed from the sample. For example, nucleic acids from organisms other than those that are detected can be present in the background in a test sample.

As used herein, the term “primer” is placed under conditions that result in the synthesis of primer extension products complementary to the nucleic acid strand (ie, in the presence of inducers such as nucleotides and DNA polymerases and at suitable temperatures and pHs). Oligonucleotides are shown whether or not naturally occurring, such as in synthetically produced or purified restriction digests, which can serve as a starting point for synthesis. The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. In the case of double strands, the primer is first processed to separate the strands and then used to prepare the expansion product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to start the synthesis of the expansion product in the presence of the inducing agent. The exact length of the primer depends on many factors, including temperature, source of primer and the use of the method.

As used herein, the term “probe” relates to whether or not it naturally occurs, such as a restriction digest produced or purified synthetically, recombinantly or by PCR amplification, which can hybridize to another oligonucleotide of interest. Oligonucleotides (ie, a series of nucleotides) are shown. The probe may be single stranded or double stranded. Probes are useful for the detection, identification and isolation of specific gene sequences. Any probe used in the present invention can be detected by any detection system, including but not limited to enzymes (eg, ELISA, as well as enzyme-based immunochemical assays), fluorescence, radioactivity and luminescence systems. It is envisioned to be labeled "reporter molecule" of. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the term “target” when used in connection with a polymerase chain reaction refers to a site of nucleic acid bound by a primer used in a polymerase chain reaction. Thus, a "target" is extracted from another nucleic acid sequence. "Segment" is defined as the site of a nucleic acid in a target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods of US Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, which are incorporated herein by reference, as known to those skilled in the art. Included are methods for increasing the concentration of segments of a target sequence in a mixture of genomic DNA without the same cloning or purification. Since the desired amplified segment of the target sequence becomes the main sequence in the mixture (in concentration), they are called "PCR amplified".

As used herein, the term “amplification reagent” refers to reagents (deoxyribonucleotide triphosphates, buffers, etc.) required for amplification, except for primers, nucleic acid templates and amplification enzymes. Typically, amplification reagents along with other reaction components are placed in a reaction vessel (test tube, microwell, etc.) and included.

By PCR, a single copy of a specific target sequence in genomic DNA can be hybridized to several different methodologies (eg, hybridization to labeled probes; avidin-enzyme conjugate detection after incorporation of biotinylation primers; ³² P into amplified segments). Amplification to detectable levels by labeled deoxynucleotide triphosphates such as incorporation of dCTP or dATP. In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with an appropriate set of primer molecules. In particular, the amplified segments produced by the PCR process itself are effective templates for subsequent PCR amplification.

As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resulting mixture of compounds after two or more cycles of PCR steps of denaturation, anneal, and expansion have been completed. This term includes the case where there is amplification of one or more segments of one or more target sequences.

As used herein, the term “RT-PCR” refers to the replication and amplification of RNA sequences. In this method, reverse transcription is coupled to PCR, the most frequently used one enzymatic method using a thermostable polymerase as described in US Pat. No. 5,322,770. In RT-PCR, RNA template is converted to cDNA due to the reverse transcriptase activity of the polymerase, which is then amplified using the polymerase's polymerization activity (ie , as in other PCR methods).

As used herein, the terms "limiting endonuclease" and "limiting enzyme" refer to bacterial enzymes that respectively cleave double stranded DNA at or near a specific nucleotide sequence.

A "restricted position" refers to a nucleotide sequence that is recognized and cleaved by a given restriction endonuclease and is often the insertion position of a DNA fragment. In certain embodiments of the invention, restriction sites are engineered into selectable markers and the 5 'and 3' ends of the DNA construct.

As used herein, the term “chromosome integration” refers to how the incoming sequence is introduced into the chromosome of a host cell. Homologous regions of the transforming DNA are aligned with the homologous regions of the chromosome. The sequence between homologous boxes is then replaced by the incoming sequence in the double crossover (ie homologous recombination). In some embodiments of the invention, the homologous sections that inactivate the chromosomal segments of the DNA construct are aligned with the lateral homologous regions of the indigenous chromosomal regions of the Bacillus chromosome. The indigenous chromosomal region is then deleted by the DNA construct within the double crossover (ie homologous recombination).

"Homologous recombination" means the exchange of DNA fragments between two DNA molecules or paired chromosomes at the same or nearly identical nucleotide sequence. In a preferred embodiment, chromosomal integration is homologous recombination.

As used herein, a "homologous sequence" is 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, with another nucleic acid or polypeptide sequence when optimally aligned for comparison. By nucleic acid or polypeptide sequence having 92%, 91%, 90%, 88%, 85%, 80%, 75% or 70% sequence identity. In some embodiments, homologous sequences have 85% to 100% sequence identity, in other embodiments 90% to 100% sequence identity, and in more preferred embodiments, 95% to 100% sequence identity.

"Amino acid" as used herein refers to a peptide or protein sequence or portion thereof. The terms "protein", "peptide" and "polypeptide" are used interchangeably.

As used herein, “protein of interest” and “polypeptide of interest” refer to a protein / polypeptide of interest and / or evaluation. In some embodiments, a “protein of interest” is a “parent protein” (ie, a starting protein). In some embodiments, the parent protein is a wild type enzyme used as a starting point for protein engineering / design. In some embodiments, the protein of interest is expressed inside the cell, while in other embodiments it is a secreted polypeptide. In particularly preferred embodiments, these enzymes include the serine proteases and metalloproteases described herein. In some embodiments, the protein of interest is a secreted polypeptide (ie, amino-terminal extension on the secreted protein) fused with a signal peptide. Nearly all secreted proteins use amino-terminal protein expansion, which plays an important role prior to the location of the precursor protein across the membrane and to target the protein. The expansion is proteolytically removed by signal peptidase during or immediately upon membrane migration.

As used herein, the term “heterologous protein” refers to a protein or polypeptide that does not occur naturally in the host cell. Examples of heterologous proteins include enzymes such as hydrolases, including proteases. In some embodiments, the gene encoding the protein is a naturally occurring gene, while in other embodiments a mutant and / or synthetic gene is used.

As used herein, “homologous protein” refers to a protein or polypeptide that is native or occurs naturally in a cell. In a preferred embodiment, the cells are Gram-positive cells, and in particularly preferred embodiments, the cells are Bacillus host cells. In alternative embodiments, homologous proteins include, but are not limited to, E. coli, Cellulomonas, Bacillus, Streptomyces, Trichoderma, and Aspergillus. It is a unique protein produced by an organism. The present invention includes host cells that produce homologous proteins by recombinant DNA technology.

As used herein, an “operon site” includes a group of contiguous genes that are transcribed from a common promoter as a single transcriptional unit and co-regulated. In some embodiments, the operon includes a regulator gene. In the most preferred embodiment, operons are used that are highly expressed as measured by RNA levels but with unknown or unnecessary functions.

As used herein, an “antibiotic site” is a site that contains one or more genes that encode antibiotic proteins.

A polynucleotide is referred to as "encoding" an RNA or polypeptide when it is in its native state or can be processed and transcribed and / or translated by methods known to those skilled in the art to produce an RNA, polypeptide or fragment thereof. Anti-sense strands of such nucleic acids are also referred to as encoding sequences.

As is known in the art, DNA can be transcribed by RNA polymerase to produce RNA, while RNA can be reverse transcribed by reverse transcriptase to produce DNA. Thus, DNA can encode RNA and vice versa.

The term "regulatory segment" or "regulatory sequence" or "expression control sequence" refers to a polynucleotide sequence of DNA operably linked to a polynucleotide sequence of DNA encoding an amino acid sequence of a polypeptide chain that affects the expression of the encoded amino acid sequence. Indicates. Regulatory sequences may inhibit, inhibit or promote the expression of operably linked polynucleotide sequences encoding amino acids.

"Host strain" or "host cell" refers to a suitable host for expression vectors comprising the DNA according to the invention.

Enzymes are “overexpressed” in a host cell when the enzyme is expressed in the cell at a level higher than that expressed in the corresponding wild type cell.

The terms "protein" and "polypeptide" are used interchangeably herein. A three digit code for an amino acid as defined according to the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. It is also understood that regression of the genetic code may allow a polypeptide to be encoded by more than one nucleotide sequence.

A "progenitor sequence" is an amino acid sequence necessary for secretion of a protease, which exists between a signal sequence and a mature protease. Cleavage of the precursor sequence will produce a mature active protease.

The term "signal sequence" or "signal peptide" refers to any sequence of nucleotides and / or amino acids that participates in the secretion of a mature or precursor form of a protein. The above definition of signal sequence is intended to include all such amino acid sequences encoded by the N-terminal portion of the protein gene, which are functional and participate in the validation of protein secretion. These, although not universal, often bind to the N-terminal portion of a protein or the N-terminal portion of a precursor protein. The signal sequence may be endogenous or exogenous. The signal sequence may be normally associated with the protein (eg protease) or may be from a gene encoding another secreted protein. One exemplary exogenous signal sequence comprises the first seven amino acid residues of the signal sequence from B. subtilis subtilisin fused to the remainder of the signal sequence of subtilisin from B. lentus (ATCC 21536).

The term “hybrid signal sequence” refers to a signal sequence obtained from an expression host in which a portion of the sequence is fused to the signal sequence of the gene to be expressed. In some embodiments, synthetic sequences are used.

The term “substantially identical signal activity” refers to signal activity as indicated by substantially equal secretion of the protease into the fermentation medium, eg, fermentation medium protease levels are secreted in the fermentation medium as provided by the signal sequence. At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% of the protease levels.

The term "mature" form of a protein or peptide refers to the final functional form of the protein or peptide. To illustrate, the mature form of the NprE protease of the present invention includes at least the amino acid sequence of SEQ ID NO: 3.

The term “precursor” form of a protein or peptide refers to a mature form of a protein having a precursor sequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a "signal" sequence operably linked to the amino terminus of the precursor sequence. The precursor may also have additional polynucleotides involved in post-translational activity (eg, polynucleotides cleaved therefrom leaving a mature form of the protein or peptide).

"Naturally occurring enzymes" and "naturally occurring proteins" refer to enzymes or proteins with unmodified amino acid sequences that match sequences found in nature. Naturally occurring enzymes include native enzymes that are naturally expressed or found in certain microorganisms.

The terms “derived from” and “obtained from” are encoded by DNA sequences isolated from such strains, as well as enzymes (eg proteases) that may or may be produced by strains of the organism in question. An enzyme produced in a host organism containing a DNA sequence is shown. In addition, the term refers to enzymes encoded by synthetic DNA sequences and / or cDNA origin and having the identifying properties of the enzyme in question.

A "derivative" within the scope of this definition generally retains the unique proteolytic activity observed in the wild type, native or parental form to the extent that the derivative is useful for similar purposes as the wild type, native or parental form. Functional enzyme derivatives include naturally occurring, synthetically or recombinantly produced peptides or peptide fragments having the general characteristics of the parent enzyme.

The term "functional derivative" refers to a derivative of nucleic acid having the functional characteristics of the nucleic acid encoding the enzyme. Functional derivatives of nucleic acids encoding enzymes provided herein include nucleic acids or fragments produced naturally, synthetically or recombinantly. Wild-type nucleic acids encoding enzymes according to the present invention include naturally occurring alleles and homologues based on degeneration of genetic codes known in the art.

The term “matching” in the context of two nucleic acid or polypeptide sequences refers to residues in the same two sequences when aligned for maximum correspondence as measured using one of the following sequence comparison or analysis algorithms.

The term “optimal alignment” refers to the alignment giving the highest% identity score.

"% Sequence identity," "% amino acid sequence identity," "% gene sequence identity," and / or "% nucleic acid / polynucleotide sequence identity" associated with two amino acids, polynucleotides, and / or gene sequences (suitably) Represents the percentage of matching residues in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in the two best aligned polypeptide sequences are identical.

Thus, the phrase “substantially identical” in the context of two nucleic acids or polypeptides is at least 70% sequence identity compared to the reference sequence using a program or algorithm (eg BLAST, ALIGN, CLUSTAL) using standard parameters, Preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98% and preferred Preferably a polynucleotide or polypeptide comprising at least 99% sequence identity. One indication that two polypeptides are substantially identical is that the first polypeptide immunologically cross reacts with the second polypeptide. Typically, different polypeptides are immunologically cross reacted by conservative amino acid substitutions. Thus, a polypeptide is substantially identical to a second polypeptide, for example if the two peptides differ only by conservative substitutions. Another indication that the two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (eg, within the medium to high stringency range).

The term “isolated” or “purified” refers to a substance removed from its original environment (eg, the natural environment if it occurs naturally). For example, the substance may be present in certain compositions at concentrations higher or lower than those naturally occurring or present in wild-type organisms or when present with components that do not normally exist upon expression from naturally-occurring or wild-type organisms. Purified. " For example, naturally occurring polynucleotides or polypeptides present in living animals are not isolated, but identical polynucleotides or polypeptides isolated from some or all of the coexisting material in nature are isolated. In some embodiments, such polynucleotides are part of a vector and / or such polynucleotides or polypeptides are part of a composition, and such vectors or compositions are not part of the natural environment. In some preferred embodiments, the nucleic acid or protein is said to be purified, for example when it appears to be essentially one band in an electrophoretic gel or blot.

When used with reference to a DNA sequence, the term “isolated” refers to a DNA sequence in a form suitable for use in genetically engineered protein production systems that is removed from its natural genetic environment and is free of other external or unwanted coding sequences. Such isolated molecules are those isolated from the natural environment and include cDNA and genomic clones. Isolated DNA molecules of the invention usually lack other genes associated with them, but may include naturally occurring 5 'and 3' untranslated sites, such as promoters and terminators. Identification of related sites will be apparent to those skilled in the art (see, eg, Dynan and Tijan, Nature 316: 774-78 [1985]). The term “isolated DNA sequence” is alternatively referred to as “cloned DNA sequence”.

When used with reference to a protein, the term “isolated” refers to a protein found under conditions other than its own environment. In a preferred form, the isolated protein is substantially free of other proteins, especially other homologous proteins. Isolated protein has a purity of greater than 10%, preferably greater than 20%, and even more preferably greater than 30%, as measured by SDS-PAGE. Additional aspects of the invention are in highly purified form (ie, greater than 40% purity, greater than 60% purity, greater than 80% purity, greater than 90% purity, greater than 95% purity, as measured by SDS-PAGE). , Greater than 97% purity, and even greater than 99% purity).

Multisite, Stratagene, La Jolla, CA)를 포함한다.As used herein, the term “combinatorial mutagenesis” refers to how a library of variants of the starting sequence is generated. In such libraries, variants contain one or several mutations selected from a predefined series of mutations. The method also provides a means for introducing random mutations that are not members of a predefined series of mutations. In some embodiments, the methods include those described in US Application 09 / 699,250, filed October 26, 2000. In alternative embodiments, combinatorial mutagenesis methods are commercially available kits (eg QUIKCHANGE).

Multisite, Stratagene, La Jolla, CA).

As used herein, the term “variant” refers to a protein derived from a precursor protein (eg “parent” protein) by insertion, substitution or deletion of one or more amino acids. In some embodiments, the variants comprise one or more modifications that include a charge change compared to the precursor protein. In some preferred embodiments, the precursor protein is a parent protein that is a wild type protein.

As used herein, the term “library of mutants” refers to a population of cells in which a majority of the genomes coincide, but contain different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved characteristics.

As used herein, the term “starting gene” refers to a gene of interest that encodes a protein of interest that is improved and / or changed using the present invention.

As used herein, the terms “multiple sequence alignments” and “MSAs” refer to multiple homologous sequences of a starting gene aligned using an algorithm (eg, Clustal W).

As used herein, the terms “consensus sequence” and “canonical sequence” refer to an archetypic amino acid sequence in which all variants of a particular protein or sequence of interest are compared. The term also refers to sequences that describe the nucleotides most often present in the DNA sequence of interest. For each position of the gene, the conserved sequence provides the most abundant amino acid at the position in MSA.

As used herein, the term “conservation mutation” refers to sequence differences between the starting gene and the conserved sequence. Conservative mutations are identified by comparing the sequences of the conserved sequence and the starting gene obtained from MSA. In some embodiments, conserved mutations are introduced into the starting gene to make them more similar to conserved sequences. Conservative mutations also include amino acid alterations in which amino acids in the starting gene are changed to amino acids more often found in MSA at positions related to the frequency of amino acids in the starting gene. Thus, the term conservative mutation includes all single amino acid changes that replace the amino acid of the starting gene with an amino acid that is more abundant than that in the MSA.

As used herein, the term “initial hit” refers to a variant identified by screening combinatorial conservative mutagenesis libraries. In a preferred embodiment, the initial hit has improved performance characteristics compared to the starting gene.

As used herein, the term “improved hit” refers to a variant identified by screening an improved combinatorial conservative mutagenesis library.

As used herein, the terms "improvement mutation" and "performance-enhancing mutation" refer to mutations that improve performance when introduced into a starting gene. In some preferred embodiments, these mutations are identified by sequencing hits identified during the screening step of such methods. In most embodiments, the mutations more often found in the hit appear to be improved mutations compared to unscreened combinatorial conservative mutagenesis libraries.

As used herein, the term “enhanced combinatorial conservative mutagenesis library” refers to a CCM library designed and constructed based on screening and / or sequencing results from initial investigations of CCM mutagenesis and screening. In some embodiments, the improved CCM library is based on the sequence of initial hits resulting from initial investigation of CCM. In additional embodiments, enhanced CCM is designed such that mutations often observed in initial hits from initial investigations of mutagenesis and screening are preferred. In some preferred embodiments, this is accomplished by omitting the primers encoding the performance-decreasing mutations or increasing the concentration of primers encoding the performance-enhancing mutations relative to other primers used in the initial CCM library.

As used herein, the term “performance-reducing mutation” refers to a mutation in a combinatorial conservative mutagenesis library that is found less frequently in hits resulting from screening compared to an unscreened combinatorial conservative mutagenesis library. In a preferred embodiment, the screening method eliminates and / or reduces the abundance of variants containing "performance-reducing mutations".

As used herein, the term “functional assay” refers to an assay that provides an indication of the activity of a protein. In a particularly preferred embodiment, the term refers to an assay system that assays for the ability of a protein to function at its usual capacity. For example, for enzymes, functional assays include measuring the effectiveness of enzymes that catalyze the reaction.

As used herein, the term "target characteristic" refers to the characteristic of the starting gene to be altered. It is not intended that the present invention be limited to any particular target property. However, in some preferred embodiments, the target property is the stability of the gene product (eg, resistance to denaturation, proteolysis or other degradation factors), and in other embodiments, the level of production in the production host is altered. Indeed, any property of the starting gene is contemplated for use in the present invention.

As used herein, the term “characteristic” or grammatical equivalent thereof, in the context of a nucleic acid, refers to any feature or attribute of the nucleic acid that can be selected or detected. These properties include properties that affect binding to polypeptides, properties endowed to cells containing specific nucleic acids, properties that affect gene transcription (e.g., promoter strength, promoter recognition, promoter regulation, enhancer function), RNA processing Properties affecting transcription (e.g., RNA splicing, RNA stability, RNA morphology and post-transcriptional modification), properties affecting transcription (e.g., level, regulation, binding, post-translational modification of mRNA for ribosomal proteins ), But is not limited to such. For example, binding sites for transcription factors, polymerases, regulatory factors, etc. of nucleic acids can be altered to produce desirable or to identify undesirable features.

As used herein, the term “property” or grammatical equivalent thereof, in the context of a polypeptide, refers to any feature or property of the polypeptide that can be selected or detected. These properties include oxidative stability, substrate specificity, catalytic activity, thermal stability, alkali stability, pH activity profile, resistance to proteolysis, K _M , k _cat , k _cat / k _M ratios, protein folding, induction of immune responses, ligands Ability to bind, ability to bind to receptors, ability to be secreted, displayed on the surface of cells, ability to form oligomers, ability to signal, ability to promote cell proliferation, ability to inhibit cell proliferation, apoptosis The ability to induce, the ability to be modified by phosphorylation or glycosylation, and the ability to treat diseases, including but not limited to.

As used herein, the term "screening" has the usual meaning in the art and is generally a multistage process. In a first step, a mutant nucleic acid or variant polypeptide therefrom is provided. In a second step, the properties of the mutant nucleic acid or variant polypeptides are measured. In a third step, the measured properties are compared with the properties of the corresponding parent nucleic acid, the properties of the corresponding naturally occurring polypeptide or the properties of the starting material (eg the initial sequence) for the production of the mutant nucleic acid.

Screening procedures for obtaining nucleic acids or proteins with altered properties will depend on the nature of the starting material, and its modifications will be apparent to those skilled in the art that the production of mutant nucleic acids is readily intended. Therefore, those skilled in the art will recognize that the present invention is not limited to any particular property to be screened, and the following list of property descriptions is described by way of example only. Screening methods for any particular property are generally described in the art. For example, binding, pH, specificity, etc. can be measured before and after mutagenesis, and changes thereof indicate alterations. Preferably, the screening is performed in a high-throughput manner including screening multiple samples simultaneously, including but not limited to assays using chips, phage display, and combinatorial substrates and / or indicators.

As used herein, in some embodiments, screening includes a selection step that enriches the variant of interest from the population of variants. Examples of these embodiments include the selection of variants that confer growth favorability to the host organism, as well as phage display or any other display method, wherein the variants are to be captured from a population of variants based on binding or catalytic properties. Can be. In a preferred embodiment, the library of variants is exposed to stress (heat, protease, denaturation), and then still intact variants are identified at screening and enriched by selection. The term is intended to include any suitable means for selection. Indeed, it is not intended that the present invention be limited to any particular screening method.

As used herein, the term “target randomization” refers to a method of generating multiple sequences of one or several positions randomized. In some embodiments, randomization is complete (ie, all four nucleotides, A, T, G, and C may occur at randomization sites). In alternative embodiments, randomization of nucleotides is limited to a subset of four nucleotides. Targeted randomization can be applied to one or several sequence codons, encoding one or several proteins of interest. When expressed, the resulting library produces a protein population in which one or more amino acid positions may contain a mixture of all 20 amino acids or a subset of amino acids, as determined by the randomization scheme of randomization codons. In some embodiments, individual members of the population resulting from target randomization differ in the number of amino acids due to the target or random insertion or deletion of the codon. In further embodiments, synthetic amino acids are included in the resulting protein populations. In some preferred embodiments, most members of the population resulting from target randomization exhibit greater sequence homology to conserved sequences than the starting gene. In some embodiments, the sequence encodes one or more proteins of interest. In alternative embodiments, the proteins have different biological functions. In some preferred embodiments, the successor sequence comprises one or more selectable markers. Such sequences may encode one or more proteins of interest. It may have other biological function (s). In many cases the successor sequence will comprise a selectable marker, such as a gene that confers resistance to antibiotics.

The terms "modified sequence" and "modified gene" are used interchangeably herein to refer to a sequence that includes the deletion, insertion, or intervention of a naturally occurring nucleic acid sequence. In some preferred embodiments, the expression product of the modified sequence is a truncated protein (eg, where the modification is deletion or intervention of the sequence). In some particularly preferred embodiments, the cleaved protein retains biological activity. In alternative embodiments, the expression product of the modified sequence is an elongated protein (eg, modifications involving insertion in nucleic acid sequences). In some embodiments, insertion results in a truncated protein (eg, when the insertion forms a stop codon). Thus, insertion can result in truncated or elongated protein as expression product.

As used herein, the terms “mutant sequence” and “mutant gene” are used interchangeably and refer to sequences in which alterations have been made in one or more codons that occur in the wild-type sequence of a host cell. Expression products of mutant sequences are proteins with altered amino acid sequences compared to wild type. Expression products may have altered functional capacity (eg, enhanced enzymatic activity).

The term "mutant primer" or "mutant oligonucleotide" (used herein interchangeably) is intended to denote an oligonucleotide composition that corresponds to a portion of the template sequence and can hybridize thereto. For mutagenic primers, the primer will not exactly match the template nucleic acid, and mismatch (es) in the primer are used to introduce the desired mutation into the nucleic acid library. As used herein, "non-mutagenic primer" or "non-mutagenic oligonucleotide" refers to an oligonucleotide composition that exactly matches a template nucleic acid. In one embodiment of the invention, only mutagenic primers are used. In another preferred embodiment of the invention, the primers are designed such that the mutagenic primers are included at one or more sites and the non-mutagenic primers included in the oligonucleotide mixture are also present. It is possible to add a mixture of mutagenic primers and non-mutagenic primers corresponding to one or more of the mutagenic primers to produce the resulting nucleic acid library with various combinatorial mutation patterns. For example, if some of the members of a mutant nucleic acid library maintain their precursor sequence at a particular location while other members prefer to be mutated at that location, the non-mutagenic primers may be non-mutated in the nucleic acid library for the indicated residue. It provides the ability to obtain specific levels of mutant members. The methods of the present invention generally use mutagenic and non-mutagenic oligonucleotides that are 10-50 bases in length, more preferably about 15-45 bases in length. However, it may be necessary to use primers shorter than 10 bases or longer than 50 bases to achieve the desired mutation results. For corresponding mutagenic and non-mutagenic primers, the lengths of the corresponding oligonucleotides need not be identical, but there are overlaps in the sites corresponding to the mutations added.

In some embodiments, the primer is added at a predefined ratio. For example, if the resulting library has a significant level of specific specific mutations, and it is desirable to have a smaller amount of different mutations at the same or different positions, the amount of primer added is adjusted to produce a library of the desired orientation. It is possible to. Alternatively, by adding less or more non-mutagenic primers, it is possible to adjust the frequency at which the corresponding mutation (s) are produced in the mutant nucleic acid library.

As used herein, the phrase “adjacent mutation” refers to a mutation present in the same oligonucleotide primer. For example, adjacent mutations may be adjacent to or near each other, but they will be introduced into the resulting mutant template nucleic acid by the same primers.

As used herein, the phrase “non-adjacent mutation” refers to a mutation present in separate oligonucleotide primers. For example, non-adjacent mutations will be introduced into the resulting mutant template nucleic acid by separately generated oligonucleotide primers.

The terms “wild type sequence,” “wild type nucleic acid sequence” and “wild type gene” are used interchangeably herein to refer to sequences that occur naturally or naturally in a host cell. In some embodiments, the wild type sequence represents the sequence of interest that is the starting point of a protein engineering project. Wild-type sequences can encode homologous or heterologous proteins. Homologous proteins are proteins that will be produced without host cell intervention. Heterologous proteins are proteins that would not be produced without host cell intervention.

The term "oxidatively stable" refers to a specific amount of enzymatic activity over a given time under proteolysis, hydrolysis, washing or other methods of the invention, such as conditions that are widely practiced during exposure to or contact with bleach or oxidant. The protease of the present invention is shown. In some embodiments, the protease is at least 50% after contact with the bleach or oxidant over a given time, for example, at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 16 minutes, 20 minutes, etc. Proteolytic activity of 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% is maintained.

The term "stable to chelating agents" refers to the specific amount of enzymatic activity over a given time under proteolysis, hydrolysis, washing or other methods of the invention, such as conditions that are widely practiced during exposure to or contact with chelating agents. The protease of the present invention is shown. In some embodiments, the protease is at least 50%, 60%, 70%, 75 after contact with the chelating agent over a given time, for example, at least 10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes, and the like. Proteolytic activity of%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% is maintained.

The terms "thermally stable" and "thermally stable" refer to proteolysis, hydrolysis, washing or other methods of the invention, for example after exposure to a temperature identified over a given time under conditions that are widely practiced during exposure to altered temperatures. Proteases of the present invention that retain specific amounts of enzyme activity are shown. Altered temperatures include increased or decreased temperatures. In some embodiments, the protease is at least 50%, 60%, 70%, 75 after exposure to altered temperatures over a given time period, such as, for example, at least 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and the like. Proteolytic activity of%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% is maintained.

In the context of proteases that are stable to oxidation, chelating agents, heat and / or pH, the term “enhanced stability” refers to higher proteolytic activity over time compared to other serine proteases (eg subtilisin proteases) and / or wild type enzymes. Indicates to keep.

In the context of proteases that are stable to oxidation, chelating agents, heat and / or pH, the term “reduced stability” refers to lower proteolysis over time compared to other serine proteases (eg subtilisin protease) and / or wild type enzymes. To maintain activity.

As used herein, the term "cleaning composition" includes, unless otherwise indicated, multipurpose or "heavy-duty" cleaning agents, especially cleaning detergents in the form of granules or powders; General-purpose cleaners in liquid, gel or paste form, in particular so-called powerful liquid forms; Liquid micro-fiber detergents; Hand dishwashers or lightweight dishwashing agents, in particular of high foaming type; Mechanical dishwashing agents including various tablet, granule, liquid and rinse aid types for home and business; Antibacterial hand wash types, wash bars, mouthwashes, denture cleaners, vehicle or carpet shampoos, bathroom cleaners; Hair shampoos and hair rinses; Liquid and fungicides, including shower gels and bubble baths and metal cleaners; Cleaning aids such as bleach additives and “stain-rods” or pretreatment types.

Unless stated otherwise, all ingredient or composition levels relate to the activity level of the ingredient or composition, and impurities (eg residual solvents or by-products) are excluded, which may be present in commercially available sources.

Enzyme component weights are based on total active protein. Unless otherwise indicated, all percentages and ratios are calculated by weight. Unless indicated otherwise, all percentages and ratios are calculated based on the total composition.

It is to be understood that all maximum numerical limits given throughout this specification include all lower numerical limits, as clearly written herein. All minimum numerical limits given throughout this specification will include all higher numerical limits as if the higher numerical limits were clearly written herein. All numerical ranges given throughout this specification will include all narrower numerical ranges falling within the broader numerical range as if the narrower numerical ranges were expressly written herein.

The term "cleaning activity" refers to the cleaning performance achieved by a protease under conditions that are widely done during proteolysis, hydrolysis, washing or other methods of the invention. In some embodiments, the cleaning performance varies after application to standard cleaning conditions and associated with enzyme sensitive stains such as, for example, grass, blood, milk, or egg proteins, as measured by various chromatographs, spectrophotometers or other quantitative methodologies. It is measured by the application of a cleaning assay. Exemplary assays include, but are not limited to, the methods included in the examples, as well as those described in WO 99/34011 and US Pat. No. 6,605,458.

The term "clean effective amount" of a protease refers to the amount of protease previously described herein that achieves the desired level of enzyme activity in a particular cleaning composition. Such effective amounts are readily identified by those skilled in the art and are based on a number of factors such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether liquid or dry (eg granules, bars) compositions are needed.

The term "cleaning aid" as used herein is selected from certain types of preferred cleaning compositions and product forms (eg liquids, granules, powders, bars, pastes, sprays, tablets, gels; or foam compositions). By any liquid, solid or gaseous substance, the substance is also preferably compatible with the protease enzyme used in the composition. In some embodiments, the granular composition is in "compressed" form, and in other embodiments, the liquid composition is in "concentrated" form.

In the context of cleaning activity, the term "improved performance" refers to an increased or greater cleaning of certain enzyme sensitive stains, such as eggs, milk, grass or blood, as measured by standard assessments after standard wash cycles and / or multiple wash cycles. Activity.

The term "reduced performance" in the context of cleaning activity refers to reduced or less cleaning activity of certain enzyme sensitive stains such as eggs, milk, grass or blood as measured by routine assessment after a standard cleaning cycle.

The term “comparative performance” in the context of cleaning activity refers to at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the cleaning activity of the comparative protease (eg, commercial protease). Cleaning performance is determined by the protease and other proteases of the present invention in various cleaning assays for enzyme sensitive stains such as blood, milk and / or ink (BMI) as measured by conventional spectrophotometric or analytical methodology after standard wash cycle conditions. Can be measured by comparing

As used herein, “low detergent concentration” systems include detergents in which less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration systems because about 667 ppm of detergent components are present in the wash water.

As used herein, “medium detergent concentration” systems include detergents in which about 800 ppm to about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems because approximately 975 ppm of detergent components are present in the wash water. Brazilian detergents typically have approximately 1500 ppm of detergent components present in the wash water.

As used herein, “high detergent concentration” systems include detergents in which more than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems because approximately 3000-8000 ppm of detergent components are present in the wash water.

"Fiber cleaning compositions" as used herein include hand washing and machines comprising compositions and laundry additive compositions suitable for use in the wetting and / or pretreatment of stained fibers (eg, fabrics, linens and other textile materials). Laundry detergent compositions are included.

“Non-fiber cleaning compositions” as used herein include non-woven (ie, fiber) surface cleaning compositions, including but not limited to dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, and personal cleansing compositions. do.

The "compressed" form of the cleaning compositions herein is best reflected by the density and by the amount of inorganic filler salt in the composition. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salt is present in significant amounts, typically 17-35% by weight of the total composition. In contrast, in compression compositions, the filler salt is present in an amount of no greater than 15% of the total composition. In some embodiments, the filler salt is present in an amount of no greater than 10% by weight of the composition, or more preferably at 5% by weight. In some embodiments, the inorganic filler salts are selected from alkali and alkaline earth metal salts of sulfates and chlorides. Preferred filler salts are sodium sulfate.

The present invention provides a method of engineering a protein such that the performance of the protein is optimized under specific environmental conditions of interest. In some embodiments, the present invention provides a method of engineering an enzyme to optimize the catalytic activity of the enzyme under certain environmental conditions. In some preferred embodiments, the present invention provides a method of engineering an enzyme such that the catalytic activity and / or stability of the enzyme under opposing environmental conditions is optimized. In some preferred embodiments, the present invention provides a method of engineering an enzyme such that the storage stability of the enzyme is optimized under particularly opposing environmental conditions. In some preferred embodiments, the present invention provides a net surface charge and / or surface of an enzyme (eg metalloprotease) such that an enzyme variant is obtained that exhibits improved performance and / or stability in detergent formulations as compared to the starting or parent enzyme. Provides a way to alter the charge distribution.

In some embodiments, the present invention provides for the enzyme to be optimized so that the catalytic activity and stability of the enzyme are simultaneously optimized under opposing environmental conditions, even if the two properties of catalyst activity and stability are inversely correlated when the effect of a single mutation is analyzed. Provides a way to manipulate. In particular, the present invention provides a method of altering the net surface charge and / or surface charge distribution of a metalloprotease such that an enzyme variant exhibiting improved performance in a detergent formulation is obtained.

The present invention provides compositions and methods comprising one or more variant neutral metalloproteases with improved washing performance and / or stability in detergent formulation (s). In some particularly preferred embodiments, the present invention provides variants of Bacillus amyloliquefaciens neutral metalloprotease. The present invention finds particular use in applications including but not limited to cleaning, bleaching and bactericides. In addition, the present invention provides a method of engineering an enzyme such that the catalytic activity of the enzyme is optimized under opposing environmental conditions. In particular, the present invention provides a method of altering the net surface charge and / or surface charge distribution of a metalloprotease such that enzyme variants exhibiting improved performance and / or stability in detergent formulations are obtained.

Many proteins and enzymes are prone to very denaturing and irreversible denaturation when stored in laundry detergents. When surfactants are classified by their ionic (electrical charge) properties in water, laundry detergents are known to contain anionic, cationic and nonionic surfactants. These components react with the surface charge of the protein molecules causing protein denaturation (eg loss of structure and function). NprE (neutral metalloprotease) has been shown to be unstable when stored in detergent formulations containing surfactants such as LAS. LAS is an anionic surfactant in which the total negative charge enhances the reaction with the positively charged side chains of amino acids located on the protein surface. This electrostatic reaction weakens or cleaves the electrostatic reaction, affecting the intrinsic stability of the protein. The destabilized protein is then released and inactivated. During the development of the present invention, it has been found that the surface charge of enzymes has a profound effect on wash performance and / or detergent stability. In addition, the residue distribution charged on the protease surface had a strong impact on wash performance and / or stability. The protein manipulation methods of the present invention optimize the net surface charge and / or surface charge distribution to effectively optimize the protease for improved performance in one or more properties in the detergent formulation.

For simplicity, in some embodiments of the present invention the method comprises the generation of a location-assessment library at many amino acid residues in the enzyme of interest, and the assay of the variant enzyme for the property of interest. This allows for optimal charge changes (relative to the parent enzyme) to the characteristic (s) of interest, as well as beneficial, neutral, and deleterious mutations. In some alternative embodiments, the charge scan of all residues to generate variants with charge changing mutations at each position, for example, mutating neutral residues to positive and / or negative charges, and reversely charged charged residues And / or neutral residues. In some further preferred embodiments, the method produces a combinatorial “charge-equilibrium” library of variants, which includes beneficial mutations that alter enzymatic charge in the desired direction, and beneficial or neutral mutations that change charge in the opposite direction, after Assaying the charge-balancing library for the property (s). Thus, the surface charge, and surface charge distribution, of the enzyme are optimized at the same time, and it is possible to identify enzymes with improvements in multiple properties.

The methods of the present invention are used to improve the performance of various classes of enzymes as well as proteases (eg amylase, cellulase, oxidase, cutinase, mannanase, pectinase, amylase, lipase, etc.). Indeed, the present invention is not intended to be limited to any particular enzyme or class of enzymes. In addition, the present invention is used for the optimization of non-enzymatic protein properties requiring specific surface charges and charge distributions (eg expression, cell-surface binding, compliance with formulations, etc.).

I. Proteases with Improved Properties Variant  produce

A large number of location-assessment libraries have been constructed for NprE, where all amino acids of the mature protein have been replaced by most other amino acids (see US Patent Application Serial Nos. 10 / 576,331 and WO 2005/052146). These libraries were screened for detergent stability and BMI cleaning performance. Screening data was then analyzed for the effect of charge alteration imparted by the mutation. Both increased stability and good BMI (blood, milk, ink) cleaning performance are desirable for engineered NprE variants, but these have initially been shown to be mutually exclusive properties. The present invention provides a means to generate more stable variants exhibiting good BMI cleaning performance.

Accordingly, the present invention provides a method for identifying mutations that give elevated stability or BMI cleaning performance without sacrificing excessively other parameters. As used herein, the phrase “overly disadvantageous” refers to protein properties with less than the desired value. The term refers to some low performing proteins with neutral mutations (less than 80% of the performance value of the parent or wild type protein); Poor performing protein with less deletion mutations (less than 50%); And essentially inactive proteins (less than 5%) with deletion mutations. In some embodiments, relative performance values are expressed as figure of merit (PI), which is the ratio of variant protein performance to parent protein performance. The charge mutation then equilibrates so that the final variant is +1 to +3 for the wild type enzyme. In addition, the present invention provides a means for selecting amino acid residues that appear to not react in the 3-D structure of an enzyme, thereby minimizing non-addition between multiple mutations.

Four NprE variants as described herein were constructed using the methods of the invention. These variants included 10 to 18 mutations. As described in more detail in the Examples, these variants exhibited increased stability and BMI cleaning performance similar to wild type enzymes.

II . Beneficial enzyme Variant  General method for producing

As described herein, the relationship between the wash performance in the BMI microsample assay and the total charge on the enzyme surface was measured. The methods of the present invention are used to improve the performance of various enzymes and proteins (eg amylase, cellulase, oxidase, cutinase, mannanase, pectinase, lipase, protease and other enzymes). Briefly, amino acid residues located on the surface of a wild-type enzyme exposed to more than about 25% of the solvent, more than about 50% of the solvent, or more than about 65% of the solvent have been identified, with each wild-type residue being numerous Position-evaluation libraries are generated that are substituted with other naturally-occurring amino acids. In some embodiments, protein engineering at the molecular surface includes replacing the neutral amino acid side chain with an acidic or basic side chain and / or replacing a positively charged side chain with a neutral or negatively charged side chain (or vice versa). In addition, to define this structure-function relationship, attention is paid to changes in the net charge of variant enzymes that result in improved wash performance in BMI. In further embodiments, once the optimal charge for a given enzyme is determined, the natural isolate is screened to identify enzyme variants with the optimal charge / charge distribution.

III . With improved properties Amylase Variant  produce

A combination of substitutions was introduced at four positions in the mature enzyme to construct a combinatorial charge library for AmyS-S242Q. The library was screened for BODIPY starch hydrolysis, rice starch microsample cleaning performance and enzyme expression. Screening data was then analyzed for the effect of charge alteration imparted by the mutation. Although both increased protein expression and good enzymatic performance are desirable for engineered AmyS-S242Q variants, these properties have yet been found to be inversely correlated. The present invention provides a means to generate more highly expressed variants that exhibit good rice starch cleaning performance. Accordingly, the present invention provides a method for identifying mutations that give elevated expression or enzymatic activity without sacrificing excessively other parameters.

Experiment

To illustrate and further illustrate certain preferred embodiments and aspects of the present invention, the following examples are provided, which are not to be construed as limiting the scope thereof.

(SEQUEST 데이터베이스 검색 프로그램, University of Washington); Npr 및 npr (중성 메탈로프로테아제 유전자); nprE 및 NprE (B. 아밀로리쿠에파시엔스 중성 메탈로프로테아제); PMN (정제된 MULTIFECT

메탈로프로테아제); MTP (마이크로타이터 플레이트); MS (질량 분광학); SRI (얼룩 제거 인자); TIGR (유전체 연구소, Rockville, MD); AATCC (미국 섬유화학 염색자 협회); Procter & Gamble (Procter & Gamble, Inc., Cincinnati, OH); Beckman (Beckman Coulter, Inc., Fullerton, CA); Amersham (Amersham Life Science, Inc. Arlington Heights, IL); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, CA); Pierce (Pierce Biotechnology, Rockford, IL); EMPA (스위스 연방재료시험 연구소, St. Gallen, Switzerland); CFT (재료 시험 센터, Vlaardingen, The Netherlands); Amicon (Amicon, Inc., Beverly, MA); ATCC (미국 미생물 보존 센터, Manassas, VA); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, NJ); Perkin-Elmer (Perkin-Elmer, Wellesley, MA); Rainin (Rainin Instrument, LLC, Woburn, MA); Eppendorf (Eppendorf AG, Hamburg, Germany); Waters (Waters, Inc., Milford, MA); Geneart (Geneart GmbH, Regensburg, Germany); Perseptive Biosystems (Perseptive Biosystems, Ramsey, MN); Molecular Probes (Molecular Probes, Eugene, OR); BioRad (BioRad, Richmond, CA); Clontech (CLONTECH Laboratories, Palo Alto, CA); Difco (Difco Laboratories, Detroit, MI); GIBCO BRL 또는 Gibco BRL (Life Technologies, Inc., Gaithersburg, MD); Epicentre (Epicentre Biotechnologies, Madison, WI); Zymo Research (Zymo Research Corp., Orange, CA); Integrated DNA Technologies (Integrated DNA Technologies, Inc., Coralville, IA): New Brunswick (New Brunswick Scientific Company, Inc., Edison, NJ); Thermoelectron (Thermoelectron Corp., Waltham, MA); BMG (BMG Labtech, GmbH, Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster, Austria); Novex (Novex, San Diego, CA); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen, Inc., Valencia, CA); Invitrogen (Invitrogen Corp., Carlsbad, CA); Sigma (Sigma Chemical Co., St. Louis, MO); DuPont Instruments (Asheville, NY); Global Medical Instrumentation 또는 GMI (Global Medical Instrumentation; Ramsey, MN); MJ Research (MJ Research, Waltham, MA); Infors (Infors AG, Bottmingen, Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Roche (Hoffmann La Roche, Inc., Nutley, NJ); Ion Beam Analysis Laboratory (Ion Bean Analysis Laboratory, The University of Surrey Ion Beam Centre (Guildford, UK); TOM (Terg-o-Meter); BMI (혈액, 우유, 잉크); BaChem (BaChem AG, Bubendorf, Switzerland); Molecular Devices (Molecular Devices, Inc., Sunnyvale, CA); MicroCal (Microcal, Inc., Northhampton, MA); Chemical Computing (Chemical Computing Corp., Montreal, Canada); NCBI (미국 생물 정보 센터); GE Healthcare (GE Healthcare, UK).The following abbreviations apply to the following experimental disclosures: ° C. (Celsius); rpm (revolutions per minute); H ₂ O (water); HCl (hydrochloric acid); aa and AA (amino acid); bp (base pair); kb (kilobase pairs); kD (kilodaltons); gm (grams); μg and ug (micrograms); mg (milligrams); ng (nanogram); μl and ul (microliters); ml (milliliters); mm (millimeters); nm (nanometer); μm and um (micrometer); M (molar); mM (millimoles); μM and uM (micromolar); U (unit); V (volts); MW (molecular weight); sec (seconds); min (minutes); hr (hours); MgCl ₂ (magnesium chloride); NaCl (sodium chloride); OD _{28 O} (optical density at 280 nm); OD ₄₀₅ (optical density at 405 nm); OD ₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); LAS (lauryl sodium sulfonate); SDS (sodium dodecyl sulfate); Tris (tris (hydroxymethyl) aminomethane); TAED (N, N, N'N'-tetraacetylethylenediamine); BES (polyester sulfone); MES (2-morpholinoethanesulfonic acid, monohydrate; fw 195.24; Sigma # M-3671); CaCl ₂ (calcium chloride, anhydrous; fw 110.99; Sigma # C-4901); DMF (N, N-dimethylformamide, fw 73.09, d = 0.95); Abz-AGLA-Nba (2-aminobenzoyl-L-alanylgylsil-L-leusil-L-alanino-4-nitrobenzylamide, fw 583.65; Bachem # H-6675, VWR Cat. No. 100040-598); SBG 1% (“Super Broth with Glucose”; 6 g Soyton [Difco], 3 g Yeast Extract, 6 g NaCl, 6 g Glucose); PH was adjusted to 7.1 with NaOH prior to sterilization using methods known in the art; w / v (weight to volume); v / v (volume to volume); Npr and npr (neutral metalloproteases); SEQUEST

(SEQUEST database search program, University of Washington); Npr and npr (neutral metalloprotease gene); nprE and NprE (B. amyloliquefaciens neutral metalloprotease); PMN (purified MULTIFECT

Metalloproteases); MTP (microtiter plate); MS (mass spectroscopy); SRI (stain removal factor); TIGR (Geogen Laboratories, Rockville, MD); AATCC (American Textile Chemical Dyeers Association); Procter & Gamble (Procter & Gamble, Inc., Cincinnati, OH); Beckman (Beckman Coulter, Inc., Fullerton, CA); Amersham (Amersham Life Science, Inc. Arlington Heights, IL); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, CA); Pierce (Pierce Biotechnology, Rockford, IL); EMPA (Swiss Federal Materials Testing Institute, St. Gallen, Switzerland); CFT (Material Testing Center, Vlaardingen, The Netherlands); Amicon (Amicon, Inc., Beverly, MA); ATCC (US Microbial Conservation Center, Manassas, VA); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, NJ); Perkin-Elmer (Perkin-Elmer, Wellesley, Mass.); Rainin (Rainin Instrument, LLC, Woburn, MA); Eppendorf (Eppendorf AG, Hamburg, Germany); Waters (Waters, Inc., Milford, Mass.); Geneart (Geneart GmbH, Regensburg, Germany); Perseptive Biosystems (Perseptive Biosystems, Ramsey, MN); Molecular Probes (Molecular Probes, Eugene, OR); BioRad (BioRad, Richmond, CA); Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Difco (Difco Laboratories, Detroit, MI); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Epicentre (Epicentre Biotechnologies, Madison, Wis.); Zymo Research (Zymo Research Corp., Orange, Calif.); Integrated DNA Technologies (Integrated DNA Technologies, Inc., Coralville, IA): New Brunswick (New Brunswick Scientific Company, Inc., Edison, NJ); Thermoelectron (Thermoelectron Corp., Waltham, Mass.); BMG (BMG Labtech, GmbH, Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster, Austria); Novex (Novex, San Diego, CA); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen, Inc., Valencia, CA); Invitrogen (Invitrogen Corp., Carlsbad, Calif.); Sigma (Sigma Chemical Co., St. Louis, Mo.); DuPont Instruments (Asheville, NY); Global Medical Instrumentation or GMI (Global Medical Instrumentation; Ramsey, MN); MJ Research (MJ Research, Waltham, Mass.); Infors (Infors AG, Bottmingen, Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Roche (Hoffmann La Roche, Inc., Nutley, NJ); Ion Beam Analysis Laboratory (Ion Bean Analysis Laboratory, The University of Surrey Ion Beam Center (Guildford, UK); TOM (Terg-o-Meter); BMI (blood, milk, ink); BaChem (BaChem AG, Bubendorf, Switzerland) ; Molecular Devices (Molecular Devices, Inc., Sunnyvale, Calif.); MicroCal (Microcal, Inc., Northhampton, Mass.); Chemical Computing (Chemical Computing Corp., Montreal, Canada); NCBI (US Biological Information Center); GE Healthcare (GE Healthcare, UK).

Example  One

Assay

The following assay was used in the examples described below. Any deviation in the protocol provided below is shown in the Examples. In these experiments, the absorbance of the product formed after completion of the reaction was measured using a spectrophotometer. The reflectance of the specimen was measured using a reflectometer.

A. Determination of Protein Content

1. For measuring protein content BCA  ( Biscinconic acid ; bicinchoninic acid ) Assay

-80). 사용된 설비는 SpectraMAX (340 유형) MTP 판독기였다. MTP는 Costar사제였다 (9017 유형). In these assays, protein concentrations in protease samples were measured on a microtiter plate (MTP) scale using the BCA (Pierce) assay. In the assay system, the following chemistry and reagent solutions were used: BCA protein assay reagent, and Pierce dilution buffer (50 mM MES, pH 6.5, 2 mM CaCl ₂ , 0.005% TWEEN

-80). The facility used was a SpectraMAX (type 340) MTP reader. MTP was manufactured by Costar (type 9017).

In the test, 200 μl BCA reagent was pipetted into each well, followed by 20 μl diluted protein. After sufficient mixing, MTP was incubated at 37 ° C. for 30 minutes. Air bubbles were removed and the optical density (OD) of the solution in the wells was read at 562 nm. To determine protein concentration, the background reading was subtracted from the sample reading. OD ₅₆₂ values were expressed against protein standards (purified protease) to generate a standard curve. Protein concentration of the sample was inferred from the standard curve.

2. for measuring protein content Bradford  ( Bradford ) Assay

In these assays, Bradford staining reagent (Quick Start) assay was used to determine protein concentration in protease samples on an MTP scale.

-80). 사용된 설비는 Biomek FX Robot (Beckman) 및 SpectraMAX (340 유형; Molecular Devices) MTP 판독기였다. MTP는 Costar사제였다 (9017 유형).In this assay system, the following chemical and reagent solutions were used: Quick Start Bradford staining reagent (BIO-RAD Cat. No. 500-0205), dilution buffer (10 mM NaCl, 0.1 mM CaC1 ₂ , 0.005% TWEEN

-80). The equipment used was a Biomek FX Robot (Beckman) and SpectraMAX (340 type; Molecular Devices) MTP reader. MTP was manufactured by Costar (type 9017).

In the test, 200 μl Bradford reagent was pipetted into each well, followed by 15 μl dilution buffer. Finally, 10 μl of filtered culture broth was added to the wells.

After sufficient mixing, MTP was incubated for at least 10 minutes at room temperature. The air bubbles were blown off and the OD of the wells was read at 595 nm. To determine protein concentration, background readings (ie, values from unvaccinated wells) were subtracted from the sample readings. The obtained OD ₅₉₅ value provides a relative measure of the protein content in the sample.

B. Microsample Assays for Testing Protease Performance

Equipment used included an Eppendorf thermal mixer and a SpectraMAX (type 340) MTP reader. MTP was manufactured by Costar (type 9017).

2X Ultra, Clean Breeze 액체 세탁 세제 (Procter & Gamble); 미국 세척 조건) Detergent preparation (TIDE

2X Ultra, Clean Breeze Liquid Laundry Detergent (Procter &Gamble); US wash conditions)

2X Ultra Clean Breeze 세제를 첨가하였다. 세제를 이전에 95℃에서 1시간 동안 열처리하여 제형에 존재하는 임의의 효소를 불활성화시켰다. 세제 용액을 15분 동안 교반하였다. 그후, 5 mM HEPES (자유산)을 첨가하고 pH를 8.2로 조정하였다.Adjust Milli-Q water to 6 gpg water hardness (Ca / Mg = 3/1), 0.78 g / 1 TIDE

2X Ultra Clean Breeze detergent was added. The detergent was previously heat treated at 95 ° C. for 1 hour to inactivate any enzymes present in the formulation. The detergent solution was stirred for 15 minutes. Then 5 mM HEPES (free acid) was added and the pH adjusted to 8.2.

Microsample

A microsample of 0.25 inch annular diameter was obtained from CFT Vlaardingen. Before cutting the specimen, the fibers (EMPA 116) were washed with water. One microsample was placed in each well of a 96-well microtiter plate.

Test Methods

Desired detergent solution was prepared as described above. After equilibrating the thermomixer at 25 ° C., 190 μl of detergent solution was added to each microsample-containing well of MTP. To the mixture, 10 μl of diluted enzyme solution was added to bring the final enzyme concentration to 1 μg / ml (measured from the BCA assay). The MTP was sealed with tape and left for 30 minutes while rotating at 1400 rpm in an incubator. After incubation under appropriate conditions, 100 μl of solution from each well was transferred to fresh MTP. Fresh MTPs containing 100 μl solution per well were read at 405 nm using an MTP SpectraMax reader. Blank controls as well as controls containing microsamples and detergents but no enzymes were also included.

Calculation of BMI Performance

The absorbance values obtained were corrected for blank test values (ie obtained after incubation of the microsample in the absence of enzyme). The resulting absorbance provided a measure of the hydrolytic activity of the enzyme tested.

안정성 검정법 C. TIDE

Stability assay

2x Ultra Clean Breeze 액체 세탁 세제의 존재 하 인큐베이션 단계 후 측정하였다. 초기 및 잔류 활성을 하기 기재된 AGLA-검정법, 형광 96-웰 플레이트 판독기, 인큐베이터/진탕기 (iEMS; Thermoelectron) 및 인큐베이터/진탕기 (Innova; New Brunswick (4230 유형)를 사용하여 측정하였다. MTP는 Costar (9017 유형) 및 Greiner (흑색 플레이트, 655076 유형)사제였다.Stability of wild type and variant proteases, 25% heat treated TIDE

Measurements were made after the incubation step in the presence of 2 × Ultra Clean Breeze liquid laundry detergent. Initial and residual activities were measured using the AGLA-assay described below, fluorescent 96-well plate reader, incubator / shaker (iEMS; Thermoelectron) and incubator / shaker (Innova; New Brunswick (4230 type)). (9017 type) and Greiner (black plate, type 655076).

Chemicals and Reagents:

In the assay system, the chemical and reagent solutions used are as follows:

2x Ultra Clean BreezeTIDE

2x Ultra Clean Breeze

2x Ultra Clean Breeze (상기와 같이 95℃에서 열처리된); 상청액 25%로 희석한 후 TIDE

농도는 27.7% 였음 ("TIDE

"로서 나타냄).125 g TIDE dissolved in a mixture of 50 g 50 mM HEPES (pH 8.2) and 275 ml water

2x Ultra Clean Breeze (heat treated at 95 ° C. as above); TIDE after dilution with 25% of the supernatant

The concentration was 27.7% ("TIDE

Represented as ").

MES dilution buffer

-80, pH 6.5; 52.6 mM MES / NaOH, 2.6 mM CaCl ₂ , 0.005% TWEEN

-80, pH 6.5

AGLA substrate

; BaChem, catalog number H-6675 or American Peptide Co., catalog number 81-0-31

AGLA substrate solution

-80, pH 6.5)에 교반하면서 부었음.; 451 mg of AGLA dissolved in 16 ml N, N-dimethylformamide; The solution was added to 304 ml of MES-buffer (52.6 mM MES / NaOH, 2.6 mM CaCl ₂ , 0.005% TWEEN

-80, pH 6.5) poured with stirring.

Test Methods:

Non-suppressed  Condition:

For the first time, 20 μl filtered culture broth was diluted with 180 μl MES dilution buffer. Thereafter, 20 μl of the diluted broth was diluted with 180 μl MES dilution buffer. 10 μl of the dilution was then diluted at 25 ° C. in a plate warmed with 190 μl AGLA-substrate solution. Any air bubbles present were blown off and the plates measured according to AGLA protease assay protocol.

Suppressed conditions:

세제 용액으로 희석하고, iEMS 진탕기에서 5분 동안 예비혼합한 후, Innova 진탕기에서 추가적으로 인큐베이션하였다. For the first time, 20 μl filtered culture broth with 180 μl TIDE

Diluted with detergent solution, premixed for 5 minutes on iEMS shaker, then further incubated on Innova shaker.

세제 용액으로 희석하고, iEMS 진탕기에서 5분 동안 예비혼합한 후, Innova 진탕기에서 추가적으로 인큐베이션하였다. 플레이트를 200 rpm에서 20℃에서 총 40분 동안 인큐베이션하였다. 그 후, 이들 용액 20 μl를 180 μl MES 희석 완충액으로 희석하고, 상기 희석물 10 μl를 25℃에서 예비가온된 플레이트에서 190 μl AGLA-기질 용액으로 희석하였다. 존재하는 어떠한 공기 방울도 불어 없애고, AGLA 프로테아제 검정법 프로토콜에 따라 플레이트를 측정하였다.Plates were incubated at 200 rpm at 32 ° C. for a total of 60 minutes. In addition, 180 μl TIDE on a 20 μl filtered culture broth

Diluted with detergent solution, premixed for 5 minutes on iEMS shaker, then further incubated on Innova shaker. The plate was incubated for 40 minutes at 20 ° C. at 200 rpm. 20 μl of these solutions were then diluted with 180 μl MES dilution buffer and 10 μl of the dilution was diluted with 190 μl AGLA-substrate solution in a pre-warmed plate at 25 ° C. Any air bubbles present were blown off and the plates measured according to AGLA protease assay protocol.

Calculation

Fluorescence measurements were taken at excitation at 350 nm and emission at 415 nm. The response rate of fluorescence increase for each well in the fluorometer software was calculated for a linear regression line of milli-RFU / min:

Percentage of Residual Activity: (Slope of Suppressed Conditions) * 100

                     (Slope of unsuppressed conditions)

D. 2- Aminobenzoyl -L- Alanylglysyl -L- Ryusil -L- Alanino -4- Nitrobenzylamide  Protease Assay ( Abz - AGLA - Nba )

The methods provided below provide a degree of technical detail that allows for reproducible protease assay data independently at time and place. While the assay can be adapted to a given experimental condition, any data obtained through the modified method must be compatible with the results produced by the original method.

Neutral metalloprotease cleaves peptide bonds between leucine and glycine of 2-aminobenzoyl-L-alanylglysil-L-leusil-L-alanino-4-nitrobenzylamide (Abz-AGLA-Nba). Free 2-aminobenzoyl-L-alanylglycine (Abz-AG) in solution has a fluorescence maximum emission at 415 nm and a maximum excitation of 340 nm. The fluorescence of Abz-AG is lost by nitrobenzylamide in the intact Abz-AGLA-Nba molecule.

In these experiments, the release of Abz-AG by protease cleavage of Abz-AGLA-Nba was monitored by fluorescence spectroscopy (excitation 340 / emission 415). The rate of appearance of Abz-AG was determined by proteolytic activity. The assay was performed under non-substrate limited initial rate conditions.

Microplate mixers with temperature control (eg Eppendorf thermal mixers) are required for reproducible assay results. The assay solution was incubated at the desired temperature (eg 25 ° C.) in a microplate mixer prior to enzyme addition. Enzyme solution was added to the plate in the mixer, mixed vigorously and quickly transferred to the plate reader.

There is a need for a fluorophotometer capable of linear regression analysis with continuous data recording, and temperature control (eg SpectraMax M5, Gemini EM, Molecular Devices). The reader was always kept at the desired temperature (eg 25 ° C.). The reader was set for peak-read fluorescence detection, excitation was set to 350 nm and emission was set to 415 nm without the use of a cutting filter. PMT was set to medium sensitivity, and five readings per well. Automatic calibration was only turned on for the first pre-read calibration. The assay was measured for 3 minutes at the reading interval minimized according to the number of wells selected to monitor. The reader was set up so that the rate of milli-RFU / min (one thousandth of relative fluorescence units per minute) was calculated. The number of readings (Vmax points) used to calculate the speed was set to a number equal to 2 minutes as measured by the reading interval (eg reading every 10 seconds used 12 points to calculate the speed). The maximum RFU was set to 50,000.

All pipetting of enzyme and substrate stock solutions was performed with positive pressure displacement pipettes (Rainin Microman). Buffers, assays and enzyme working solutions were pipetted from tubes, reagent reservoirs or storage microplates by single or multi-channel air substitution pipettes (Rainin LTS). Repeater pipettes (Eppendorf) are useful for transferring assay solution to microplate wells to minimize loss of reagents when only a few wells are used. Automated pipetting instruments such as Beckman FX or Cybio Cybi-well are also useful for transferring enzyme solutions from functional storage microplates to assay microplates for simultaneous inoculation of the entire microplate.

Reagents and Solutions:

52.6 mM MES / NaOH, 2.6 mM CaCl ₂ , pH 6.5-MES buffer

; MES acid (10.28 g) and 292 mg anhydrous CaCl ₂ were dissolved in about 90O mL purified water. The solution was titrated to pH 6.5 with NaOH (at 25 ° C. or with a temperature adjusted pH probe). pH-adjusted buffer was prepared in 1 L total volume. The final solution was filtered through a 0.22 μm sterile filter and left at room temperature.

Abz-AGLA-Nba 48 mM—Abz-AGLA-Nba stock in DMF

; About 28 mg of Abz-AGLA-Nba was placed in a small tube. Dissolved in DMF (volume varies depending on aggregated Abz-AGLA-Nba) and vortexed for several minutes. The solution was protected from light and stored at room temperature.

50 mM MES, 2.5 mM CaCl ₂ , 5% DMF, 2.4 mM Abz-AGLA-Nba pH 6.5-Assay Solution

; 1 mL Abz-AGLA-Nba stock was added to 19 mL MES buffer and vortexed. The solution was protected from light and stored at room temperature.

50 mM MES, 2.5 mM CaCl ₂ , pH 6.5-enzyme dilution buffer

; 5 mL purified water was added to 95 mL MES buffer to prepare the buffer.

50 mM MES, 2.5 mM CaCl ₂ , 5% DMF, pH 6.5-substrate dilution buffer

; 5 mL pure DMF was added to 95 mL MES buffer. The buffer was used to measure motor parameters.

Enzyme solution

중성 프로테아제 (야생형 NprE)를 6 ppm (6 μg/mL) 미만의 농도로 희석하였다. 연속 희석이 바람직하였다. 용액은 실온에서 1시간 동안 안정하였으나, 더 긴 저장 기간을 위해서, 용액을 얼음에서 유지시켰다.; The enzyme stock solution was diluted to a concentration of about 1 ppm (1 μg / mL) using enzyme dilution buffer. MULTIFECT

Neutral protease (wild type NprE) was diluted to a concentration of less than 6 ppm (6 μg / mL). Serial dilution is preferred. The solution was stable for 1 hour at room temperature, but for a longer storage period the solution was kept on ice.

step

First, all buffers, stocks and working solutions were prepared. Unless indicated otherwise, each enzyme dilution was assayed in triplicate. If not entirely, enzymatic solution storage microplates were arranged in the entire longitudinal column starting from the left side of the plate (to be accommodated in the plate reader). The corresponding assay plate was set similarly. The microplate fluorometer was set up as previously described.

First, 200 μL aliquots of the assay solution were placed in wells of a 96-well microplate. Plates were protected from light and incubated for 10 minutes at 25 ° C. in a controlled microplate mixer. 10 uL of the functional enzyme from the storage microplate was transferred to the assay microplate in a mixer to initiate the assay. Optionally, 96-well pipetting heads were used, or in some experiments, 8-well multi-channel pipettes were used to transfer from the leftmost column first. The solution was mixed vigorously for 15 seconds (900 rpm on an Eppendorf heat mixer). Immediately, the assay microplates were transferred to a microplate fluorometer and fluorescence measurement recording was started at excitation at 350 nm and emission at 415 nm. The response rate of fluorescence increase for each well in the fluorometer software was calculated for a linear regression line of milli-RFU / min. In some experiments, the second plate was placed in a microplate mixer for temperature equilibration while reading the first plate.

The initial rate was linear with product concentration up to 0.3 mM product (ie, free 2-aminobenzoyl fluorescence), which was approximately in solution starting at 2.3 mM Abz-AGLA-Nba, with a background fluorescence of about 22,000 RFU. Corresponding to 50,000 RFU. Abz-AGLA-Nba was dissolved in DMF and used on the day of preparation.

Example  2

B. In subtilis NprE  Protease Production

In the above examples, the experiments performed to generate NprE protease in B. subtilis are described. In particular, methods are provided for transforming plasmid pUBnprE into B. subtilis. Transformation was performed as known in the art (see, eg, WO 02/14490 and US Patent Application Serial No. 11 / 581,102). DNA sequences encoding the NprE precursor protein (nprE leader from B. amyloliquefaciens, nprE precursor and nprE mature DNA sequences) are provided below.

In this sequence, the bold portion represents DNA encoding the mature NprE protease, the standard font represents the leader sequence (nprE leader) and the underlined portion represents the precursor sequence (nprE precursor). The amino acid sequences provided below (NprE leader, NprE precursor and NprE mature DNA sequence) (SEQ ID NO: 2) correspond to the full length NprE protein. In this sequence, the underlined portion represents the precursor sequence and the bold portion represents the mature NprE protease.

The mature NprE sequence is described as SEQ ID NO: 3. The sequence was used as the basis for preparing the variant libraries described herein.

The pUBnprE expression vector was constructed by amplifying the nprE gene from the chromosomal DNA of B. amyloliquefaciens by PCR using the following two specific primers:

Oligo AB 1740: CTGCAGGAATTCAGATCTTAACATTTTTCCCCTATCATTTTTCCCG (SEQ ID NO: 4); And

Oligo B 1741: GGATCCAAGCTTCCCGGGAAAAGACATATATGATCATGGTGAAGCC (SEQ ID NO: 5).

PCR was performed in a thermocycler using Phusion highly compatible DNA polymerase (Finnzymes). PCR mixtures were 10 μl 5x buffer (Finnzymes Phusion), 1 μl 10 mM dNTP, 1.5 μl DMSO, 1 μl of each primer, 1 μl Finnzymes Phusion DNA polymerase, 1 μl chromosomal DNA solution 50 ng / μl, 34.5 μl MilliQ Water. The following PCR protocol was used: 1) 30 seconds at 98 ° C .; 2) 10 seconds at 98 ° C .; 3) 20 seconds at 55 ° C .; 4) 1 minute at 72 ° C .; 5) 25 cycles of steps 2 to 4; And 6) 5 minutes at 72 ° C.

This resulted in a 1.9 kb DNA fragment, which was digested using BglII and BclI DNA restriction enzymes. The multicopy Bacillus vector pUB110 (see eg Gryczan, J Bacteriol, 134: 318-329 [1978]) was digested with BamHI. PCR fragment x BglII x BclI was then linked in the pUB110 x BamHI vector to form a pUBnprE expression vector.

pUBnprE was transformed against B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE: :( xylR, pxylA-comK) strains B. Transformation into subtilis was directed to WO 02/14490. Selective growth of B. subtilis transformants with pUBnprE vector was obtained in shake flasks containing 20 mg / L neomycin, with 25 ml MBD medium (medium as defined by MOPS). NH ₄ Cl ₂ , FeSO ₄ and CaCl ₂ are excluded from the base medium, 3 mM K ₂ HPO ₄ is used, and the base medium is supplemented with 60 mM urea, 75 g / L glucose and 1% soytone. MBD media were prepared essentially as known in the art (see Neidhardt et al., J Bacteriol, 119: 736-747 [1974].) In addition, micronutrients were added to 1 L, 400 mg FeSO ₄ .7H _2. O, 100 mg MnSO ₄ ㆍ H ₂ O, 100 mg ZnSO ₄ ㆍ 7H ₂ O, 50 mg CuCl ₂ ㆍ 2H ₂ O, 100 mg CoCl ₂ ㆍ 6H ₂ O, 100 mg NaMoO ₄ ㆍ 2 Prepared as a 100 X stock solution containing H ₂ O, 100 mg Na ₂ B ₄ O ₇ 10H ₂ O, 10 ml of 1 M CaCl ₂ , and 10 ml of 0.5 M sodium citrate The culture was incubator / Incubation was incubated for 3 days at 37 ° C. The culture resulted in production of secreted NprE protease with proteolytic activity as demonstrated by protease assay NuPage Novex 10% Bis-Tris. Gel analysis was performed using a gel (Invitrogen, Cat. No. NP0301BOX) To prepare a sample for analysis, two volumes of the supernatant were prepared using 1 volume 1 M HCl, 1 volume 4 × LDS sample buffer (Invitrogen, catalog # NP0007), And 1% PMSF (20 mg / ml) and then heated at 70 ° C. for 10 minutes. 25 μL of each sample was then loaded onto the gel with 10 μL of SeeBlue + 2 pre-stained protein standard (Invitrogen, Cat. No. LC5925). The results clearly demonstrate that the nprE cloning strategy described in the above examples is suitable for the generation of active NprE in B. subtilis.

Example  3

Location evaluation library ( SEL ) Generation

In this embodiment, the method used in the construction of the nprE SEL is described.

The pUBnprE vector containing the nprE expression cassette described above served as template DNA. The vector contains a unique BglII restriction site, which was used in the location assessment library construct. Briefly, to construct an nprE position assessment library, two mutagenesis PCRs for introducing a mutated codon of interest into a mature nprE DNA sequence, and a construct for a pUBnprE expression vector comprising the desired mutated codons in the mature nprE sequence Three PCR reactions were performed, including a third PCR used to fuse two mutagenesis PCRs.

The mutagenesis method was based on a codon-specific mutation approach, where the specifically designed triple DNA sequence NNS (N = A), which corresponds to the sequence of codons to be mutated and ensures random incorporation of nucleotides in specific nprE mature codons The generation of all possible mutations simultaneously in specific DNA triplets using forward and reverse oligonucleotide primers having 25 to 45 nucleotides in length, containing C, T or G; and S = C or G). Was performed. The numbers listed in the primer names correspond to specific nprE mature codon positions. The positions evaluated included: 4, 12, 13, 14, 23, 24, 33, 45, 46, 47, 49, 50, 54, 58, 59, 60, 65, 66, 87, 90, 96 , 97, 100, 186, 196, 211, 214, 228 and 280. An exemplary list of primer sequences is described in US patent application Ser. No. 11 / 581,102.

Two additional primers used to construct the location assessment library contained a portion of the pUBnprE DNA sequence flanking the BglII restriction site, along with the BglII restriction site. These primers were prepared by Invitrogen (50 nmole scale, decontaminated): pUB-BglII-FW GTCAGTCAGATCTTCCTTCAGGTTATGACC (SEQ ID NO: 6); And pUB-BglII-RV GTCTCGAAGATCTGATTGCTTAACTGCTTC (SEQ ID NO: 7).

Two primary PCR amplifications using pUB-BglII-FW primers and specific nprE reverse mutagenesis primers initiated the construction of each SEL. For secondary PCR, pUB-BglII-RV primers and specific nprE forward mutagenesis primers (same nprE mature codon position for forward and reverse mutagenesis primers) were used.

The introduction of mutations in mature nprE sequences was performed using Phusion high conformance DNA polymerase (Finnzymes; catalog number F-530L). All PCRs were performed according to the Finnzymes protocol supplied with the polymerase. PCR conditions for the first PCR are as follows:

1st PCR 1:

pUB-BglII-FW primers and specific NPRE reverse mutagenesis primers-both 1 μL (10 μM);

1st PCR 2:

pUB-BglII-RV primers and specific NPRE forward mutagenesis primers-both 1 μL (10 μM);

With this,

5 x Phusion HF Buffer 1 O μL

1 μL of 10 mM dNTP mixture

Phusion DNA Polymerase 0.75 μL (2 units / μL)

DMSO, 100% 1 μL

1 μL of pUBnprE template DNA (0.1 to 1 ng / μL)

50 μL or less of distilled, autoclaved water.

PCR programs were: 30 seconds at 98 ° C., 30 × (10 seconds at 98 ° C., 20 seconds at 55 ° C., 1.5 minutes at 72 ° C.) and 5 minutes at 72 ° C., at PTC-200 Peltier Thermal Cycler (MJ Research). Was performed. PCR experiments resulted in two fragments of about 2-3 kB, which had about 30 nucleotide base overlaps around the NprE mature codon of interest. These two aforementioned fragments and the forward and reverse BglII primers were used to fuse the fragments in the third PCR reaction. Fusion PCR reactions were run in the following solutions: pUB-BglII-FW primers and pUB-BglII-RV primers-both 1 μL (10 μM),

With this,

5 x Phusion HF Buffer 10 μL

1 μL of 10 mM dNTP mixture

Phusion DNA Polymerase 0.75 μL (2 units / μL)

DMSO, 100% 1 μL

1 μL of the first PCR 1 reaction mixture

1 μL of the first PCR 2 reaction mixture

50 μL or less of distilled, autoclaved water.

The PCR fusion program was as follows: 30 seconds at 98 ° C., 30 × (10 seconds at 98 ° C., 20 seconds at 55 ° C., 2 minutes 40 seconds at 72 ° C.) and 5 minutes at 72 ° C., between PTC-200 Peltier thermals. Done by MJ Research.

PCR 정제 키트 (Qiagen, 카탈로그 번호 28106)를 사용하여 증폭된 선형 6.5 Kb 절편을 정제하고, 하기와 같이 BglII 제한 효소로 소화시켜 융합 절편의 양쪽 면 상에 점착 말단을 생성시켰다: QIAQUICK

Amplified linear 6.5 Kb fragments were purified using a PCR purification kit (Qiagen, Cat. No. 28106) and digested with BglII restriction enzymes to produce sticky ends on both sides of the fusion fragment as follows:

35 μL purified linear DNA fragment

3 완충액 (Invitrogen)4 μL REACT

3 buffer (Invitrogen)

1 μL BglII, 10 units / ml (Invitrogen)

Reaction conditions: 1 hour, 30 ° C.

PCR 정제 키트 (Qiagen, 카탈로그 번호 28106)를 사용하여 정제된 절편의, 하기와 같은 연결은 원하는 돌연변이를 함유하는 환형 및 다중 결합성 DNA를 야기한다:BglII Digested, and QIAQUICK

The following linkage of sections purified using PCR Purification Kit (Qiagen, Cat. No. 28106) results in circular and multiple binding DNAs containing the desired mutations:

30 μL of purified BglII digested DNA fragment

8 μL T4 DNA ligase buffer (Invitrogen Cat. No. 46300-018)

-1 μL T4 DNA ligase, 1 unit / μL (Invitrogen Cat. No. 15224-017)

Reaction conditions: 16-20 hours, 16 degreeC.

The ligation mixture was then transformed into B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE: :( xylR, pxylA-comK) strains. / 14490. As for each library, 96 single colonies were selected for sequencing (BaseClear) and screening purposes and grown in MOPS medium with neomycin and 1.25 g / L yeast extract, respectively. The library of contained up to 19 nprE position-specific variants.

B. subtilis SEL transformants in 96 well MTP were grown in MBD medium with 20 mg / L neomycin and 1.25 g / L yeast extract for 68 hours at 37 ° C. to generate variants.

Example  4

돌연변이유발을 통한 변이체 프로테아제의 생성 QUIKCHANGE

Generation of variant proteases through mutagenesis

In the above examples, although the methods provided herein are suitable for the production of SELs of other enzymes of interest (eg Asp), alternative methods for generating nprE SELs are described. As in Example 3 above, the pUBnprE vector containing the nprE expression cassette served as a template DNA source for the production of nprE SEL and NprE variants. The main difference between the two methods is that the method requires amplification of the whole vector using complementary position-directed mutagenic primers.

matter:

Bacillus strains containing the pUBnprE vector

Qiagen Plasmid Midi Kit (Qiagen Catalog No. 12143)

Ready-Lyse Resozyme (Epicentre Catalog No. R 1802M)

dam methylase kits (New England Biolabs Catalog No. M0222L)

Zymoclean Gel DNA Recovery Kit (Zymo Research Catalog No. D4001)

nprE position-directed mutagenic primers, 100 nmole scale, 5 'phosphorylated, PAGE purified (Integrated DNA Technologies)

다중 위치-지정 돌연변이유발 키트 (Stratagene 카탈로그 번호 200514) QUIKCHANGE

Multiple Position-Specific Mutagenesis Kit (Stratagene Catalog No. 200514)

MJ Research PTC-200 Peltier Thermal Cycler (Bio-Rad Laboratories)

1.2% Agarose E-gel (Invitrogen Catalog No. G5018-01)

TempliPhi Amplification Kit (GE Healthcare Catalog No. 25-6400-10)

Competent B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE: :( xylR, pxylA-comK).

Way:

다중 위치-지정 돌연변이유발 키트로 공급됨) (즉, 1.5 μl DpnI 제한 효소를 각각의 튜브에 첨가하고 3시간 동안 37℃에서 인큐베이션하였음)한 후; PCR 반응이 작용하였는지, 및 모체 주형이 분해되었는지 확실히 하기 위해 2 μl의 DpnI-소화된 PCR 반응물을 1.2% E-gel에서 분석하였다. 이후 nprE 다중 변이체의 라이브러리 크기를 증가시키기 위해, 제조사의 프로토콜을 사용하여 대량의 DNA가 생성되도록 TempliPhi 회전 원형 증폭을 사용하였다 (즉, ~11 μl 총 반응물에 대해, 1 μl DpnI 처리된 QuikChange 다중 위치-지정 돌연변이유발 PCR, 5 μl TempliPhi 샘플 완충액, 5 μl TempliPhi 반응 완충액 및 0.2 μl TempliPhi 효소 혼합물; 3시간 동안 3O℃에서 인큐베이션; 200 μl 증류된, 오토클레이브된 물을 첨가하여 TempliPhi 반응물을 희석하고, 짧게 볼텍싱). 그 후, 1.5 μl의 희석된 TempliPhi 물질을 적격 B. 서브틸리스 세포 내에 형질전환시키고, LA + 10 ppm 네오마이신 + 1.6 % 스킴 밀크 플레이트를 사용하여 nprE 다중 변이체를 선택하였다. 콜로니를 고른 후 서열화하여 상이한 nprE 변이체 라이브러리 조합을 확인하였다.To obtain a pUBnprE plasmid containing one mutation (identified by nprE SEL screening as described in Example 4 and US Patent Application Serial No. 11 / 581,102 above), a single colony of each Bacillus strain of interest was 5 ml LB + 10 ppm neomycin tubes (eg starter culture) were used to inoculate. Cultures were grown at 37 ° C. with shaking at 225 rpm for 6 hours. Thereafter, 100 ml of fresh LB + 10 ppm neomycin was inoculated with 1 ml of seed culture. The culture was grown overnight at 37 ° C. with shaking at 225 rpm. After this incubation, cell pellets were harvested by centrifugation sufficiently to provide cell pellets. Cell pellets were resuspended in 10 ml buffer P1 (Qiagen plasmid midi kit). 10 μl of Ready-Lyse lysozyme was then added to the resuspended cell pellet and incubated at 37 ° C. for 30 minutes. The Qiagen plasmid midi kit protocol was continued using 10 ml of buffers P2 and P3 to account for the increased volume of cell culture. After each pUBnprE plasmid containing a single nprE mutation was isolated from Bacillus, the concentration of each plasmid was measured. The plasmid was then dam methylated using the dam methylase kit (New England Biolabs) according to the manufacturer's instructions so that about 2 μg of each pUBnprE plasmid per tube was methylated. The dam-methylated pUBnprE plasmid was purified and concentrated using a Zymoclean Gel DNA recovery kit. The dam-methylated pUBnprE plasmids were then quantified and diluted to a working concentration of 50 ng / μl for each. Mixed site-directed mutagenic primers were prepared separately for each reaction. For example, using the pUBnprE T14R plasmid as a template source, the mixed position-directed mutagenic primer tubes were prepared with 10 μl of nprE-S23R, 10 μl nprE-G24R, 10 μl nprE-N46K and 10 μl nprE-T54R (all Primers each at 10 μΜ). 1 μl dam methylated pUBnprE plasmid (containing one mutation) (50 ng / μl), 2 μl nprE position-directed mutagenic primer (10 μM, according to manufacturer's instructions, for example to provide 25 μl total reaction mixture) ), 2.5 μl 10 × QuikChange Multiple Reaction Buffers, 1 μl dNTP mixture, 1 μl QuikChange Multiple Enzyme Formulation (2.5 U / μl) and 17.5 μl Distilled, Autoclaved Water) QuikChange Multiple Site-Specific Mutagenesis Kit (Stratagene) PCR reaction was performed. The nprE variant library was amplified using the following conditions: 1 minute at 95 ° C. (first cycle only), then 1 minute at 95 ° C., 1 minute at 55 ° C., 13.5 minutes at 65 ° C., and 29 cycles repeated. The reaction product was stored at 4 ° C. overnight. Subsequently, the reaction mixture was subjected to DpnI digestion (QUIKCHANGE) using the manufacturer's protocol so that the parent pUB-nprE plasmid was digested.

Supplied in a multiple site-directed mutagenesis kit) (ie 1.5 μl DpnI restriction enzyme was added to each tube and incubated at 37 ° C. for 3 hours); 2 μl of DpnI-digested PCR reactions were analyzed on 1.2% E-gel to ensure that the PCR reaction worked and the parental template was degraded. Then, to increase the library size of the nprE multiple variant, TempliPhi rotational circular amplification was used to generate large amounts of DNA using the manufacturer's protocol (i.e., 1 μl DpnI treated QuikChange multiple sites for ˜11 μl total reactants). Designate mutagenesis PCR, 5 μl TempliPhi sample buffer, 5 μl TempliPhi reaction buffer and 0.2 μl TempliPhi enzyme mixture; incubate at 3O ° C. for 3 hours; dilute the TempliPhi reaction by adding 200 μl distilled, autoclaved water, Short vortexing). Thereafter, 1.5 μl of diluted TempliPhi material was transformed into eligible B. subtilis cells and nprE multiple variants were selected using LA + 10 ppm neomycin + 1.6% scheme milk plates. Colonies were picked and sequenced to identify different nprE variant library combinations.

Table 4-1 provides primer names and the sequences used in these experiments. All primers were synthesized by Integrated DNA Technologies (100 nmole scale, 5′-phosphorylated, PAGE purified). Additional mutagenesis primers are described in US patent application Ser. No. 11 / 581,102. The evaluated positions include: 4, 12, 13, 23, 45, 49, 50, 54, 59, 60, 65, 82, 90, 110, 119, 128, 129, 130, 135, 136, 137 , 138, 139, 140, 151, 152, 155, 179, 190, 197, 198, 199, 204, 205, 214, 216, 217, 218, 219, 220, 221, 222, 224, 243, 244, 260 , 261, 263, 265, 269, 273, 282, 285, 286, 289, 293, 296, 297 and 299.

Example  5

Variant  Expression, Fermentation, Purification and Analysis of Proteases

This example describes the method used to express, ferment and purify the protease of the transformed B. subtilis of the previous example.

원심분리기 모델 RC5B). 분비된 중성 메탈로프로테아제를 배양 유체로부터 단리하고, BES (폴리에테르설폰) 10 kDa 절삭을 갖는 Amicon filter system 8400을 사용하여 약 10배 농축하였다. Recombinant Bacillus subtilis were cultivated by conventional batch fermentation in nutrient medium. One glycerol vial of B. subtilis containing B. amyloliquefaciens neutral metalloprotease was used to inoculate 600 ml of SBG 1% medium containing 200 mg / L chloramphenicol. After growing embryos at 37 ° C. for 36-48 hours, the culture fluid was recovered by centrifugation at 12,000 rpm as is known in the art. The method was performed in duplicate. Final enzyme concentrations obtained during the 48 hour incubation ranged from about 1.4 to 2 g / L. After 36 hours incubation at 37 ° C., the fermentation broth was recovered and centrifuged at 12,000 rpm (SORVALL

Centrifuge model RC5B). Secreted neutral metalloproteases were isolated from the culture fluid and concentrated about 10-fold using an Amicon filter system 8400 with BES (polyethersulfone) 10 kDa cutting.

SDS-PAGE (Novex)를 사용하여 평가하였다. 분획을 함유하는 중성 프로테아제를 이후 모 았다. pH를 5.8로 조정하기 전에 3:1 비율의 염화칼슘염 및 염화아연염을 첨가하였다. Perceptive Biosystems BIOCAD

Vision (GMI)을 단백질 정제에 사용하였다.Concentrated supernatant was dialyzed against 25 mM MES buffer, pH 5.4, containing 10 mM NaCl overnight at 4 ° C. The dialysate was then loaded into the cation-exchange column Poros HS20 (total volume ˜83 mL; binding volume ˜4.5 g protein / mL column; waters) as described below. The column was pre-equilibrated with 25 mM MES buffer at pH 5.4 containing 10 mM NaCl. Thereafter, about 200-300 mL of sample was loaded into the column. Bound proteins were eluted using a pH gradient of 5.4 to 6.2 over a 10-column volume of MES buffer. The eluate of the protein was pH 5.8 to 6.0, proteolytic activity and 10% (w / v) NUPAGE as described above

Evaluation was made using SDS-PAGE (Novex). Neutral proteases containing fractions were then collected. Calcium chloride and zinc chloride salts were added in a 3: 1 ratio before adjusting the pH to 5.8. Perceptive Biosystems BIOCAD

Vision (GMI) was used for protein purification.

SDS-PAGE를 사용하여 평가된 정제된 단백질은 95% 초과의 순도로 균질한 것으로 측정되었다. 전형적으로, 기질, N-숙시닐-L-Ala-L-Ala-L-Pro-L-Phe-p-니트로아닐리드 (Bachem)로의 표준 프로테아제 검정법을 사용하여 평가되는 경우, 정제된 제조물은 사소한 세린 프로테아제 활성을 나타내었다. 10 mM CaCl₂ 및 0.005% TWEEN

-80을 함유하는 pH 8.5의 100 mM 트리스-HCl 완충액을 사용하여 상기 검정법을 마이크로타이터 플레이트 (MTP) 포맷 (96 웰)에서 수행하였다. DMSO (디메틸설폭시드) 중 160 mM 저장액 (100 mg/ml)을 제조하고 상기 저장액을 CaCl₂ 및 0.005% TWEEN

-80을 함유하는 트리스-HCl 완충액으로 100배 희석하여, 기질 (p-AAPF NA)을 제조하였다. 그 후 10 μL의 희석된 프로테아제 용액 (10 mM CaCl₂ 및 0.005 % TWEEN

-80을 함유하는 pH 8.5의 100 mM 트리스-HCl 완충액을 사용하여 희석물을 제조하였음)을 190 μL 1 mg/ml p-AAPF NA 용액에 첨가하였다. 검정물을 5분 동안 혼합하고 410 nm에서 운동 변화를 2 내지 5분에 걸쳐 판독하였다. 반응의 기울기를 측정하고 세린 프로테아제 활성량의 표현으로서 사용하였다. 1 mM 염화아연, 4 mM 염화칼슘 및 40% 프로필렌 글리콜을 함유하는 25 mM MES 완충액을 사용하여 저장을 위해 단백질을 제형화하였다.10% (w / v) NUPAGE

Purified protein evaluated using SDS-PAGE was determined to be homogeneous with greater than 95% purity. Typically, when assessed using a standard protease assay with the substrate, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Bachem), the purified preparations may be Protease activity was shown. 10 mM CaCl ₂ and 0.005% TWEEN

The assay was performed in microtiter plate (MTP) format (96 wells) using 100 mM Tris-HCl buffer at pH 8.5 containing -80. Prepare 160 mM stocks (100 mg / ml) in DMSO (dimethylsulfoxide) and store the stocks with CaCl ₂ and 0.005% TWEEN.

Substrates were diluted 100-fold with Tris-HCl buffer containing -80 to prepare substrates (p-AAPF NA). Then 10 μL of diluted protease solution (10 mM CaCl ₂ and 0.005% TWEEN

Dilutions were made using 100 mM Tris-HCl buffer at pH 8.5 containing −80) was added to 190 μL 1 mg / ml p-AAPF NA solution. The assay was mixed for 5 minutes and the kinetic change at 410 nm was read over 2-5 minutes. The slope of the reaction was measured and used as a representation of the amount of serine protease activity. Proteins were formulated for storage using 25 mM MES buffer containing 1 mM zinc chloride, 4 mM calcium chloride and 40% propylene glycol.

Example  6

Equilibrium for Protease Activity and Stability Mutant  effect

This example describes multi-substituted protease variants engineered to optimize two opposing enzyme properties. Table 6-1 below shows how the charge change was calculated.

As measured during the development of the present invention, the median stability decreased with increasing positive charge. However, BMI cleaning performance increased with increasing positive charge. Under test conditions, an optimum BMI cleaning performance was obtained with a charge change of about +1.

Improved stability and BMI cleaning performance are desirable in engineered variants of NprE. However, these characteristics are in a seemingly opposite characteristic. As measured using the methods of the present invention during the development of the present invention, selective combination of single mutations can produce more stable variants without severely impairing BMI cleaning performance. The strategy described herein provides for multi-substituted NprE variants that improve the first property (eg, stability as the primary property) while not improving or sacrificing the second property (eg, BMI cleaning performance as the secondary property). It was used successfully to create. In particular, the following criteria were used to select substitutions of interest. Mutations were selected that provide elevated detergent stability or BMI cleaning performance without excessively sacrificing other parameters. In addition, the charge mutations are in equilibrium, with the final variant being +1 to +3 relative to the wild type enzyme. In addition, amino acid residues have not been shown to react in the 3-D structure such that non-additionality between multiple mutations is minimized.

During the development of the present invention, four variants each containing 10 to 18 substitutions were constructed. These variants are shown in Table 6-3. Importantly, these multi-substituted variants have increased detergent stability and similar cleaning performance compared to wild type enzymes. This was accomplished by introducing an equilibrium positive charge performance mutation that does not excessively affect stability, with negative and neutral charge stability mutations that do not significantly harm BMI cleaning performance. Additional sets of variant pairs were constructed. The first of each pair has a stabilizing negative charge mutation with reduced BMI cleaning performance and the second of each pair has a compensating positive charge mutation with restored BMI cleaning performance while maintaining stability above wild-type levels. Cleaning performance and stability values for these variants are also shown in Table 6-4.

Example  7

Amylase  Equilibrium for Activity and Expression Mutant  effect

This example illustrates that two opposing enzyme properties can be optimized simultaneously due to the introduction of multiple amino acid substitutions.

In this example, Bacillus stearomophilus alpha amylase (also referred to herein as AmyS), AmyS truncated form thereof, which is a mutant in B. subtilis (S242Q with 29 amino acid deletions, also referred to herein as S242Q) Experiments performed to generate variants are described. Transformation was performed as known in the art (see eg WO 02/14490). Briefly, genes encoding maternal amylase were cloned into pHPLT expression vectors containing LAT promoter (PLAT), sequences encoding LAT signal peptide (preLAT), and then PstI and HpaI restriction sites for cloning.

B. Steareromophilus AmyS - S242Q CCL Generation of

AmyS-S242Q plasmid DNA was isolated from transformed B. subtilis strains (genotypes: ΔaprE, ΔnprE, amyE: :( xylRPxylAcomK-phleo) and sent to DNA2.0 Inc. as a template for CCL construction. DNA242 Inc. (Mountain View, CA) was asked for the generation of a positional library at each of four positions in S242Q (S242Q) amylase.Variants were supplied as glycerol stocks in 96-well plates. AmyS S242Q combinatorial charge library was designed by identifying four residues: Gln-97, Gln 319, Gln 358 and Gln 443. All combinations of three possibilities (wild-type, arginine or aspartic acid) at each position were made to four positions, 81-membered CCL was generated.

The amino acid sequence of mature cleaved S242Q amylase with substituted amino acids represented in italics was used as the basis for making the variant libraries described herein:

Amylase  Expression-2 ml  scale

B. subtilis clones containing AmyS, S242Q or AmyTS23t expression vectors were prepared in 96-well culture plates (BD, 353075) containing 150 μl of LB medium + 10 μg / ml neomycin from glycerol stock. Duplicated in a 96-well replicator and grown overnight at 37 ° C., 220 rpm in a humidified confined space. 100 μl aliquots from overnight cultures were used to inoculate 2000 μl defined medium + 10 μg / ml neomycin in 5 ml plastic culture tubes. The culture medium was a rich semi-defined medium based on MOPS buffer, with urea as the main nitrogen source, glucose as the main carbon source, and supplemented with 1% soyton and 5 mM calcium for robust cell growth. Culture tubes were incubated at 37 ° C., 250 rpm for 72 hours. After the incubation, the culture broth was centrifuged at 3000 x g for 10 minutes. The supernatant solution was transferred to a 15 ml polypropylene conical tube and 80 μL of each sample was dispensed into 96 well plates for protein quantification.

By antibody titration Amylase  Concentration measurement

As described herein, alpha-amylase concentration and specific activity were measured by titration with inhibitory polyclonal antibodies. Polyclonal antibodies elevated against Bacillus stearomophilus alpha-amylase (AmyS) have been found to potently inhibit alpha-amylase and AmyS from Bacillus species TS23 (eg binding results in a linear titration of activity loss). Hard enough to be). Therefore, the antibody can be used to measure antibody concentration, which is then used to calculate specific activity. Briefly, the amount of enzyme inhibition produced by various known concentrations of antibodies is measured. From this information, the concentration of antibody required for complete inhibition is estimated, which is equivalent to the enzyme concentration in the sample. Alpha-amylase activity and inhibition were measured using a fluorescence BODIPY-starch assay. The buffer was 50 mM MOPS, pH 7.0, containing 0.005% Tween-80.

Polyclonal antibodies directed against purified AmyS were elevated in rabbits and purified by standard methods. Inhibition of a sample of known specific activity of AmyS was measured to determine the "obvious concentration" value based on the experiment of the antibody stock solution. The antibody samples were then used to measure the specific activity and concentration of AmyS and TS23t variants. These values were used to generate normalized 96-well enzyme storage plates in which all variants were diluted to normal concentrations.

Amylase  For measuring activity Bodipy Starch assay

울트라 아밀라아제 검정 키트 (E33651, Invitrogen)를 사용하여 Bodipy-전분 검정법을 수행하였다. pH 4.0에서 100 μL의 5O mM 나트륨 아세테이트 완충액 중 동결건조된 기질을 함유하는 바이알의 내용물을 용해하여 DQ 전분 기질의 1 mg/mL 저장 용액을 제조하였다. 바이알을 약 20초 동안 볼텍싱하고, 암 환경 하에서, 용해될 때까지 비정기적으로 혼합하면서 실온에 두었다. 900 μL의 검정 완충액 (50 mM 나트륨 아세테이트와 2.6 mM CaCl₂ pH 5.8)을 첨가하고, 바이알을 약 20초 동안 볼텍싱하였다. 기질 용액을 사용 준비가 될 때까지 암 환경 하에 실온에서, 또는 4℃에서 보관하였다. 검정을 위해, 검정 완충액 중 1 mg/mL 기질 용액으로부터 DQ 기질의 작용 용액 100 μg/mL를 제조하였다. 190 μL의 100 μg/mL 기질 용액을 96-웰 평평한 바닥 마이크로타이터 플레이트에서의 각각의 웰에 첨가하였다. 10 μL의 효소 샘플을 웰에 첨가하고, 열혼합기를 사용하여 800 rpm에서 30초 동안 혼합하였다. 완충액 및 기질만을 함유하는 공시험 샘플 (효소 공시험 샘플 아님)을 검정법에 포함시켰다. 형광 마이크로타이터 플레이트 판독기에서 25℃에서 5분 동안 형광 세기의 변화 속도를 측정하였다 (여기: 485 nm, 방출: 520 nm).EnzChek

Bodipy-starch assays were performed using the Ultra Amylase Assay Kit (E33651, Invitrogen). A 1 mg / mL stock solution of DQ starch substrate was prepared by dissolving the contents of the vial containing the lyophilized substrate in 100 μL of 50 mM sodium acetate buffer at pH 4.0. The vial was vortexed for about 20 seconds and, under dark environment, left at room temperature with occasional mixing until dissolved. 900 μL of assay buffer (50 mM sodium acetate and 2.6 mM CaCl ₂ pH 5.8) was added and the vials were vortexed for about 20 seconds. The substrate solution was stored at room temperature or at 4 ° C. under dark environment until ready for use. For the assay, 100 μg / mL of the working solution of DQ substrate was prepared from 1 mg / mL substrate solution in assay buffer. 190 μL of 100 μg / mL substrate solution was added to each well in a 96-well flat bottom microtiter plate. 10 μL of enzyme sample was added to the wells and mixed for 30 seconds at 800 rpm using a heat mixer. Blank test samples (not enzyme blank samples) containing only buffer and substrate were included in the assay. The rate of change in fluorescence intensity was measured for 5 minutes at 25 ° C. in a fluorescence microtiter plate reader (excitation: 485 nm, emission: 520 nm).

Alpha- Amylase  Combination

Amylase variants were incubated for 30 minutes under standard washing conditions with or without CS-28 rice starch microsample. The amount of free enzyme was measured by the BODIPY-starch assay. The fraction of enzyme bound to the microsample was calculated as follows: bound fraction = (activity of the enzyme in the absence of the sample-activity of the enzyme in the presence of the sample) / (activity of the enzyme in the absence of the sample)

result

As measured during the development of the present invention, the median expression of AmyS-242Q decreased with increasing positive charge. However, specific BODIPY starch hydrolysis increased with increasing positive charge. Improved recombinant amylase expression and starch hydrolysis are preferred in engineered variants of AmyS-242Q that are suitable for, for example, starch liquefaction or detergent cleaning in the fuel ethanol industry. However, these characteristics are clearly opposite characteristics. As measured using the methods of the invention during the development of the present invention, selective combination of single mutations can produce more highly expressed amylase variants without severely impairing starch hydrolysis. The strategy described herein is a multi-substituted AmyS-242Q that does not improve or sacrifice a second property (eg starch hydrolysis as a secondary property) while improving the first property (eg expression as a primary property). It has been successfully used to generate and select variants.

In addition, contrary to the median expression of AmyS-242Q variant, rice starch microsample washing increased with increasing positive charge. Improved recombinant amylase expression and cleaning performance are desirable in engineered variants of AmyS-242Q. However, these properties are also clearly opposite properties. As determined using the methods of the present invention during the development of the present invention, selective combination of single mutations can produce more highly expressed amylase variants without severely impairing wash performance. The strategy described herein provides a multi-substituted AmyS that does not improve or sacrifice the second property (eg, rice starch microsample cleaning as the second property) while improving the first property (eg, expression as a primary property). It has been successfully used to generate and select -242Q variants.

In particular, 80-membered AmyS-S242Q Charge Combination Library (CCL) comprising variants having a combination of one to four substitutions of charged residues was tested for shaking tube expression, BODIPY-starch hydrolysis and rice starch cleaning activity. . AmyS-S242Q successes are shown in Tables 7-1 and 7-2. Importantly, the multi-substituted variants of Table 7-1 have equivalent or improved expression and equivalent or improved BODIPY-starch hydrolysis compared to the parent enzyme. Similarly, the multi-substituted variants of Table 7-2 have equivalent or improved expression and equivalent or improved rice starch washing activity compared to the parent enzyme.

In short, because enzyme activity and enzyme production have different charge dependencies (see FIGS. 2A, 2B, 3A and 3B), they are inversely correlated (see FIGS. 1A and 1B). However, there are many variants with both improved expression and activity, and libraries are identified in this way to identify them.

Although proven to be amylase, the method also applies to other classes of enzymes such as proteases, lipases, cellulases, transferases and pectinase. In addition, any combination of two or more properties can be analyzed simultaneously (eg expression, activity, binding, thermal stability, detergent and / or chelant stability).

All patents and publications mentioned herein are at the level of those skilled in the art to which this invention belongs. Those skilled in the art readily recognize that the present invention is well adapted to carry out the stated objects and obtain the stated objects and advantages, as well as those inherent in the invention. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be apparent to those skilled in the art that various permutations and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention described in the description herein may suitably be practiced without any element (s), limitation (s) not specifically disclosed herein. The terms and expressions used are for the purpose of description and not of limitation, and are not intended to use these terms and expressions, except for any equivalents of the features shown or described, but are not intended to be within the scope of the claimed invention. It is appreciated that various modifications are possible. Thus, while the invention has been specifically disclosed by its preferred embodiments and any features, variations and changes in the concepts described herein may be reclassified by those skilled in the art, and such variations and changes are as defined herein. It should be understood that it is considered to be within the same scope of the present invention.

The present invention has been described broadly and generically herein. Each of the narrower species and lower genus groups included within the generic disclosure also form part of the present invention. This includes the generic name description of the present invention with the proviso or negative limitation of removing any subject from the generic name, whether or not the deleted material is specifically mentioned herein.

<110> Danisco US Inc., Genencor Division          Aehle, Wolfgang          Cascao-Pereira, Luis Gustavo          Kellis Jr., James T.          Shaw, Andrew <120> Methods for Improving Protein Properties <130> 30973WO-2 <140> PCT / US2008 / 007114 <141> 2008-06-06 <150> US 60 / 933,307 <151> 2007-06-06 <150> US 60 / 933,331 <151> 2007-06-06 <150> US 60 / 933,312 <151> 2007-06-06 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 1566 <212> DNA <213> Bacillus amyloliquefaciens <400> 1 gtgggtttag gtaagaaatt gtctgttgct gtcgccgctt cctttatgag tttaaccatc 60 agtctgccgg gtgttcaggc cgctgagaat cctcagctta aagaaaacct gacgaatttt 120 gtaccgaagc attctttggt gcaatcagaa ttgccttctg tcagtgacaa agctatcaag 180 caatacttga aacaaaacgg caaagtcttt aaaggcaatc cttctgaaag attgaagctg 240 attgaccaaa cgaccgatga tctcggctac aagcacttcc gttatgtgcc tgtcgtaaac 300 ggtgtgcctg tgaaagactc tcaagtcatt attcacgtcg ataaatccaa caacgtctat 360 gcgattaacg gtgaattaaa caacgatgtt tccgccaaaa cggcaaacag caaaaaatta 420 tctgcaaatc aggcgctgga tcatgcttat aaagcgatcg gcaaatcacc tgaagccgtt 480 tctaacggaa ccgttgcaaa caaaaacaaa gccgagctga aagcagcagc cacaaaagac 540 ggcaaatacc gcctcgccta tgatgtaacc atccgctaca tcgaaccgga acctgcaaac 600 tgggaagtaa ccgttgatgc ggaaacagga aaaatcctga aaaagcaaaa caaagtggag 660 catgccgcca caaccggaac aggtacgact cttaaaggaa aaacggtctc attaaatatt 720 tcttctgaaa gcggcaaata tgtgctgcgc gatctttcta aacctaccgg aacacaaatt 780 attacgtacg atctgcaaaa ccgcgagtat aacctgccgg gcacactcgt atccagcacc 840 acaaaccagt ttacaacttc ttctcagcgc gctgccgttg atgcgcatta caacctcggc 900 aaagtgtatg attatttcta tcagaagttt aatcgcaaca gctacgacaa taaaggcggc 960 aagatcgtat cctccgttca ttacggcagc agatacaata acgcagcctg gatcggcgac 1020 caaatgattt acggtgacgg cgacggttca ttcttctcac ctctttccgg ttcaatggac 1080 gtaaccgctc atgaaatgac acatggcgtt acacaggaaa cagccaacct gaactacgaa 1140 aatcagccgg gcgctttaaa cgaatccttc tctgatgtat tcgggtactt caacgatact 1200 gaggactggg atatcggtga agatattacg gtcagccagc cggctctccg cagcttatcc 1260 aatccgacaa aatacggaca gcctgataat ttcaaaaatt acaaaaacct tccgaacact 1320 gatgccggcg actacggcgg cgtgcataca aacagcggaa tcccgaacaa agccgcttac 1380 aatacgatta caaaaatcgg cgtgaacaaa gcggagcaga tttactatcg tgctctgacg 1440 gtatacctca ctccgtcatc aacttttaaa gatgcaaaag ccgctttgat tcaatctgcg 1500 cgggaccttt acggctctca agatgctgca agcgtagaag ctgcctggaa tgcagtcgga 1560 ttgtaa 1566 <210> 2 <211> 521 <212> PRT <213> Bacillus amyloliquefaciens <400> 2 Met Gly Leu Gly Lys Lys Leu Ser Val Ala Val Ala Ala Ser Phe Met   1 5 10 15 Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ala Glu Asn Pro Gln              20 25 30 Leu Lys Glu Asn Leu Thr Asn Phe Val Pro Lys His Ser Leu Val Gln          35 40 45 Ser Glu Leu Pro Ser Val Ser Asp Lys Ala Ile Lys Gln Tyr Leu Lys      50 55 60 Gln Asn Gly Lys Val Phe Lys Gly Asn Pro Ser Glu Arg Leu Lys Leu  65 70 75 80 Ile Asp Gln Thr Thr Asp Asp Leu Gly Tyr Lys His Phe Arg Tyr Val                  85 90 95 Pro Val Val Asn Gly Val Pro Val Lys Asp Ser Gln Val Ile Ile His             100 105 110 Val Asp Lys Ser Asn Asn Val Tyr Ala Ile Asn Gly Glu Leu Asn Asn         115 120 125 Asp Val Ser Ala Lys Thr Ala Asn Ser Lys Lys Leu Ser Ala Asn Gln     130 135 140 Ala Leu Asp His Ala Tyr Lys Ala Ile Gly Lys Ser Pro Glu Ala Val 145 150 155 160 Ser Asn Gly Thr Val Ala Asn Lys Asn Lys Ala Glu Leu Lys Ala Ala                 165 170 175 Ala Thr Lys Asp Gly Lys Tyr Arg Leu Ala Tyr Asp Val Thr Ile Arg             180 185 190 Tyr Ile Glu Pro Glu Pro Ala Asn Trp Glu Val Thr Val Asp Ala Glu         195 200 205 Thr Gly Lys Ile Leu Lys Lys Gln Asn Lys Val Glu His Ala Ala Thr     210 215 220 Thr Gly Thr Gly Thr Thr Leu Lys Gly Lys Thr Val Ser Leu Asn Ile 225 230 235 240 Ser Ser Glu Ser Gly Lys Tyr Val Leu Arg Asp Leu Ser Lys Pro Thr                 245 250 255 Gly Thr Gln Ile Ile Thr Tyr Asp Leu Gln Asn Arg Glu Tyr Asn Leu             260 265 270 Pro Gly Thr Leu Val Ser Ser Thr Thr Asn Gln Phe Thr Thr Ser Ser         275 280 285 Gln Arg Ala Ala Val Asp Ala His Tyr Asn Leu Gly Lys Val Tyr Asp     290 295 300 Tyr Phe Tyr Gln Lys Phe Asn Arg Asn Ser Tyr Asp Asn Lys Gly Gly 305 310 315 320 Lys Ile Val Ser Ser Val His Tyr Gly Ser Arg Tyr Asn Asn Ala Ala                 325 330 335 Trp Ile Gly Asp Gln Met Ile Tyr Gly Asp Gly Asp Gly Ser Phe Phe             340 345 350 Ser Pro Leu Ser Gly Ser Met Asp Val Thr Ala His Glu Met Thr His         355 360 365 Gly Val Thr Gln Glu Thr Ala Asn Leu Asn Tyr Glu Asn Gln Pro Gly     370 375 380 Ala Leu Asn Glu Ser Phe Ser Asp Val Phe Gly Tyr Phe Asn Asp Thr 385 390 395 400 Glu Asp Trp Asp Ile Gly Glu Asp Ile Thr Val Ser Gln Pro Ala Leu                 405 410 415 Arg Ser Leu Ser Asn Pro Thr Lys Tyr Gly Gln Pro Asp Asn Phe Lys             420 425 430 Asn Tyr Lys Asn Leu Pro Asn Thr Asp Ala Gly Asp Tyr Gly Gly Val         435 440 445 His Thr Asn Ser Gly Ile Pro Asn Lys Ala Ala Tyr Asn Thr Ile Thr     450 455 460 Lys Ile Gly Val Asn Lys Ala Glu Gln Ile Tyr Tyr Arg Ala Leu Thr 465 470 475 480 Val Tyr Leu Thr Pro Ser Ser Thr Phe Lys Asp Ala Lys Ala Ala Leu                 485 490 495 Ile Gln Ser Ala Arg Asp Leu Tyr Gly Ser Gln Asp Ala Ala Ser Val             500 505 510 Glu Ala Ala Trp Asn Ala Val Gly Leu         515 520 <210> 3 <211> 300 <212> PRT <213> Bacillus amyloliquefaciens <400> 3 Ala Ala Thr Thr Gly Thr Gly Thr Thr Leu Lys Gly Lys Thr Val Ser   1 5 10 15 Leu Asn Ile Ser Ser Glu Ser Gly Lys Tyr Val Leu Arg Asp Leu Ser              20 25 30 Lys Pro Thr Gly Thr Gln Ile Ile Thr Tyr Asp Leu Gln Asn Arg Glu          35 40 45 Tyr Asn Leu Pro Gly Thr Leu Val Ser Ser Thr Thr Asn Gln Phe Thr      50 55 60 Thr Ser Ser Gln Arg Ala Ala Val Asp Ala His Tyr Asn Leu Gly Lys  65 70 75 80 Val Tyr Asp Tyr Phe Tyr Gln Lys Phe Asn Arg Asn Ser Tyr Asp Asn                  85 90 95 Lys Gly Gly Lys Ile Val Ser Ser Val His Tyr Gly Ser Arg Tyr Asn             100 105 110 Asn Ala Ala Trp Ile Gly Asp Gln Met Ile Tyr Gly Asp Gly Asp Gly         115 120 125 Ser Phe Phe Ser Pro Leu Ser Gly Ser Met Asp Val Thr Ala His Glu     130 135 140 Met Thr His Gly Val Thr Gln Glu Thr Ala Asn Leu Asn Tyr Glu Asn 145 150 155 160 Gln Pro Gly Ala Leu Asn Glu Ser Phe Ser Asp Val Phe Gly Tyr Phe                 165 170 175 Asn Asp Thr Glu Asp Trp Asp Ile Gly Glu Asp Ile Thr Val Ser Gln             180 185 190 Pro Ala Leu Arg Ser Leu Ser Asn Pro Thr Lys Tyr Gly Gln Pro Asp         195 200 205 Asn Phe Lys Asn Tyr Lys Asn Leu Pro Asn Thr Asp Ala Gly Asp Tyr     210 215 220 Gly Gly Val His Thr Asn Ser Gly Ile Pro Asn Lys Ala Ala Tyr Asn 225 230 235 240 Thr Ile Thr Lys Ile Gly Val Asn Lys Ala Glu Gln Ile Tyr Tyr Arg                 245 250 255 Ala Leu Thr Val Tyr Leu Thr Pro Ser Ser Thr Phe Lys Asp Ala Lys             260 265 270 Ala Ala Leu Ile Gln Ser Ala Arg Asp Leu Tyr Gly Ser Gln Asp Ala         275 280 285 Ala Ser Val Glu Ala Ala Trp Asn Ala Val Gly Leu     290 295 300 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 4 ctgcaggaat tcagatctta acatttttcc cctatcattt ttcccg 46 <210> 5 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 5 ggatccaagc ttcccgggaa aagacatata tgatcatggt gaagcc 46 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 6 gtcagtcaga tcttccttca ggttatgacc 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 7 gtctcgaaga tctgattgct taactgcttc 30 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 8 ggtacgactc ttaaaggaaa aagagtctca ttaaatattt cttctgaaag 50 <210> 9 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 9 gtctcattaa atatttcttc tgaaagaggc aaatatgtgc tgcgcgatc 49 <210> 10 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 10 ctcattaaat atttcttctg aaagcagagg caaatatgtg ctgcgcgatc 50 <210> 11 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 11 cacaaattat tacgtacgat ctgcaaaaac gcgagtataa cctgc 45 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer <400> 12 gtataacctg ccgggcagac tcgtatccag caccacaaac cag 43 <210> 13 <211> 486 <212> PRT <213> Artificial Sequence <220> <223> synthetic amylase <400> 13 Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu   1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn              20 25 30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys          35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp      50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr  65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met                  85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly             100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln         115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe     130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr                 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu             180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His         195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn     210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Gln Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly                 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys             260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp         275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala     290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln                 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala             340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp         355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile     370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val                 405 410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro             420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val         435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser     450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr                 485 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic reagent <400> 14 Ala Gly Leu Ala   One <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic substrate <400> 15 Ala Ala Pro Phe   One  

Claims (32)

A method of producing an improved protein variant, comprising the following steps in a feasible order: a) testing a plurality of mono-substituted protein variants in a first test of a first property and a second test of a second property, wherein the properties of the parent protein are given a value of 1.0 in each test, The first or second property has a value of greater than 1.0 and the first or second property that is excessively disadvantageous has a value of less than about 0.80; b) identifying a substitution in one or more of the mono-substituted protein variants that are associated with the first advantageous property and not the second property that are excessively disadvantageous; c) identifying a substitution in at least one of the mono-substituted protein variants associated with the second advantageous property and not overly unfavorable the first property; d) introducing the substitution from step b) and the substitution from step c) into a protein to obtain a multi-substituted protein variant, wherein said multi-substituted protein variant is an improved protein variant. The method of claim 1 further comprising the following steps: e) testing the multi-substituted protein variants in the first and second trials, wherein the improved protein variants achieve values greater than 1.0 in both the first and second trials, or 1.0 in the first trial. Achieve values exceeding and achieving a value between 0.80 and 1.0 in the second test. 3. The method of claim 2, wherein said parent protein is an enzyme and said improved protein variants are enzymes. 4. The method of claim 3, wherein said enzyme is selected from proteases, amylases, cellulases, polyesterases, esterases, lipases, cutinases, pectinase, oxidases, transferases and catalases. The method of claim 4, wherein the enzyme is a protease or amylase. 4. The method of claim 3, wherein the first and second properties of interest are substrate binding, enzyme inhibition, expression, stability in detergents, thermal stability, reaction rate, degree of reaction, thermal activity, starch liquefaction, biomass degradation, glycosylation, ester valence A process comprising at least two of the group consisting of degradation, enzymatic bleaching, washing performance and fabric modification. 4. The method of claim 3, further comprising generating an improved protein variant. 4. The method of claim 3, wherein the first and second characteristics are negatively correlated. 4. The method of claim 3, wherein said advantageous first or second property has a value greater than about 1.2. 4. The method of claim 3, wherein said excessively adverse first or second characteristic has a value of less than about 0.60. The method of claim 10, wherein said excessively adverse first or second characteristic has a value of less than about 0.40. 4. The method of claim 3 wherein the first property is stability and the second property is wash performance. 13. The method of claim 12, wherein said stability comprises stability in detergent compositions and said cleaning performance comprises blood milk ink (BMI) cleaning performance. The method of claim 13, wherein said cleaning performance is tested in a liquid detergent composition or powder having a pH of about 5 to about 12. The method of claim 13, wherein said washing performance is tested in a cold water liquid detergent having a basic pH. 4. The method of claim 3 wherein the first property is protein expression and the second property is enzymatic activity. 5. The method of claim 4, wherein said protease is selected from neutral metalloproteases and serine proteases. The method of claim 13, wherein said serine protease is subtilisin. 18. The method of claim 17, wherein the neutral metalloprotease is a neutral metalloprotease obtained from a member of the family Bacillaceae. 5. The method of claim 4, wherein said amylase is an alpha amylase obtained from a member of the family Basilaceae. 4. The method of claim 3, wherein at least one of the substitutions comprises a net charge change of 0, -1 or -2 with respect to the parent enzyme. 4. The method of claim 3, wherein at least one of the substitutions comprises a net charge change of +1 or +2 relative to the parent enzyme. 4. The method of claim 3, wherein at least one of the substitutions comprises a net charge change of 0, -1 or -2 with respect to the parent enzyme. 4. The method of claim 3, wherein at least one of the substitutions comprises a net charge change of +1 or +2 relative to the parent enzyme. 4. The method of claim 3, wherein said improved enzyme variant has a net charge change of +1 or +2 relative to the parent enzyme. 4. The method of claim 3 wherein the substitution is in position at the parent enzyme having greater than about 25% solvent accessible surface (SAS). The method of claim 3, wherein the substitution is in position at the parent enzyme with greater than about 50% or greater than about 65% solvent accessible surface (SAS). 4. The method of claim 3, wherein said parent enzyme is a wild type enzyme. A cleaning composition comprising the improved protein variant produced according to the method of claim 3. An isolated neutral metalloprotease variant having an amino acid sequence comprising one or more substitutions of amino acids made at a position equivalent to a position in the neutral metalloprotease comprising the amino acid sequence set forth in SEQ ID NO: 3. 31. The isolated neutral metalloprotease of claim 30, wherein said one or more substitutions are made at positions equivalent to position 83 of the amino acid sequence set forth in SEQ ID NO: 3. 32. The isolated neutral metalloprotease of claim 31, wherein said substitution is L83K.

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